BR122021002178B1 - Use of pyrimidinyl tyrosine kinase inhibitor compounds - Google Patents

Abstract

The present invention provides compounds and compositions thereof useful as Bruton's tyrosine kinase inhibitors and having desirable characteristics therefor.

Description

Cross-Reference to Related Orders

[0001] The present application claims priority to the U.S. provisional application. serial number 61/657,360, filed June 8, 2012, all contents of which are hereby incorporated by reference.

Background of the Invention

[0002] Tec kinases are non-receptor tyrosine kinases including: Tec (tyrosine kinase expressed in hepatocellular carcinoma), Btk (Bruton's tyrosine kinase), Itk (interleukin-2 (IL-2) inducible T cell kinase); also known as Emt or Tsk), Rlk (resting lymphocyte kinase; also known as Txk), Lck (lymphocyte-specific protein tyrosine kinase), and Bmx (bone marrow tyrosine kinase gene on the X chromosome; also known as Etk) ). These kinases are mainly expressed in hematopoietic cells, although the expression of Bmx and Tec has been detected in endothelial and hepatic cells. Tec kinases (Itk, Rlk and Tec) are expressed on T cells and are all activated downstream of the T cell receptor (TCR). Btk is a downstream mediator of the B cell receptor (BCR) that signals who is involved in regulating B cell activation, proliferation, and differentiation. More specifically, Btk contains a PH domain that binds phosphatidylinositol (3,4, 5)-trisphosphate (PIP3). Binding to PIP3 induces Btk to phosphorylate phospholipase C (PLCY), which in turn hydrolyzes PIP2 to produce two secondary messengers, inositol triphosphate (IP3) and diacylglycerol (DAG), which activates the PKC protein kinase , which then induces additional signaling from B cells. Mutations that inactivate the enzymatic activity of Btk result in XLA syndrome (X-linked agammaglobulinemia), a primary immunodeficiency. Given the critical roles that Tec kinases play in B cell and T cell signaling, Tec are targets of interest in autoimmune disorders.

[0003] Consequently, there is a great need in the art for effective inhibitors of Tec kinases, such as Btk. The present invention meets these and other needs.

Summary of the Invention

[0004] In certain embodiments, the present invention provides a compound of formula I:

[0005] or a pharmaceutically acceptable salt thereof, wherein R1 and Ring A are as defined and described herein. These compounds are inhibitors of the Tec kinase family, including Btk. In this regard, compounds are provided that can be used in a variety of methods including screening and in vitro activity assays, as well as preclinical, clinical and therapeutic in vivo situations, as described in detail herein.

[0006] In certain embodiments, the present invention provides pharmaceutical formulations comprising the compounds provided.

[0007] In certain embodiments, the present invention provides a method of decreasing the enzymatic activity of Btk. In some embodiments, these methods include contacting the Btk with an effective amount of a Btk inhibitor.

[0008] In certain embodiments, the present invention provides a method of treating a disorder responsive to Btk inhibition in a subject in need. These disorders and methods are described in detail in this document.

Detailed Description of Certain Modalities

[0009] In certain embodiments, the present invention provides a compound of formula I:

[00010] or a pharmaceutically acceptable salt thereof;

[00011] where:

[00012] each R1 is independently hydrogen, an optionally substituted C1-6 aliphatic group, in optionally substituted 37-membered monocyclic heterocyclic group, or an optionally substituted heterocyclylalkyl group having 3-7 carbon atoms and 1-3 heteroatoms independently selected from nitrogen , oxygen or sulfur;

[00013] or two R1 groups are taken together with their intervening atoms to form an optionally substituted saturated or partially unsaturated 3-7 membered monocyclic heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen or sulfur;

[00014] wherein the optionally substituted groups may be substituted with halogen, -NO2, -CN, -OR, -SR, -N(R)2, -C(O)R, -CO2R, -N(R)C (O)OR, -C(O)N(R)2, -OC(O)R, -N(R)C(O)R, -S(O)R, -S(O)2R, or - S(O)2N(R)2;

[00015] each R is independently hydrogen or C1-6 aliphatic;

[00016] or two R groups bonded to the same nitrogen are taken together with their intervening atoms to form an optionally substituted saturated or partially unsaturated 3-7 membered monocyclic heterocyclic ring having 1-2 heteroatoms, wherein any second heteroatom is independently selected of nitrogen, oxygen or sulfur; R2

[00017] Ring A is ;

[00018] R2 is -Cl or -F; and

[00019] R3 is -CF3, -OCF3, or -F.

Definitions

[00020] The compounds of this invention include those generally described above, and are further illustrated by the classes, subclasses, and species disclosed herein. As used in this document, the following definitions shall apply unless stated otherwise. For the purposes of this invention, chemical elements are identified according to the Periodic Table of Elements, CAS version, Handbook of Chemistry and Physics, 75th Ed. Additionally, general principles of organic chemistry are described in "Organic Chemistry", Thomas Sorrell, University Science Books, Sausalito: 1999, and "March's Advanced Organic Chemistry", 5th Ed., Ed.: Smith, M.B. and March, J., John Wiley & Sons, New York: 2001, all contents of which are hereby incorporated by reference.

[00021] Abbreviations used in this document have their conventional meaning in chemical and biological techniques. The chemical structures and formulas depicted in this document are constructed according to standard rules of chemical valence known in the chemical arts.

[00022] The term "aliphatic" or "aliphatic group", as used herein, means a straight (i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain that is fully saturated or that contains one or more unsaturation units, or a monocyclic hydrocarbon or bicyclic hydrocarbon that is fully saturated or contains one or more units of unsaturation, but which is not aromatic (also referred to herein as "carbocycle", "cycloaliphatic" or "cycloalkyl"), which has a single point of attachment to the rest of the molecule. Unless otherwise specified, aliphatic groups contain 1-6 aliphatic carbon atoms. In some embodiments, the aliphatic groups contain 1-5 aliphatic carbon atoms. In some embodiments, the aliphatic groups contain 1-4 aliphatic carbon atoms. In some embodiments, the aliphatic groups contain 1-3 aliphatic carbon atoms, and in still other embodiments, the aliphatic groups contain 1-2 aliphatic carbon atoms. Suitable aliphatic groups include, but are not limited to, linear or branched, substituted or unsubstituted alkyl, alkenyl, alkynyl groups and hybrids thereof, such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl, or (cycloalkyl)alkenyl.

[00023] As used herein, the term "cycloaliphatic" (or "carbocycle or "cycloalkyl") refers to a C3-C6 monocyclic hydrocarbon that is fully saturated or contains one or more units of unsaturation, but which is not aromatic, which has a single point of attachment to the rest of the molecule.

[00024] As used herein, the terms "heterocycle", "heterocyclyl" and "heterocyclic ring" are used interchangeably and refer to a stable 3- to 7-membered monocyclic heterocyclic moiety that is saturated or partially unsaturated, and that has , in addition to carbon atoms, one or more, preferably from one to four, heteroatoms, as defined above. When used in reference to an atom of a heterocycle, the term "nitrogen" includes a substituted nitrogen. For example, in a saturated or partially unsaturated ring having 0-3 heteroatoms selected from oxygen, sulfur or nitrogen, the nitrogen can be N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl) , or +NR (as in N-substituted pyrrolidinyl). The term "heterocyclylalkyl" refers to an alkyl group substituted by a heterocyclyl, wherein the alkyl and heterocyclyl moieties are independently optionally substituted.

[00025] A heterocyclic ring may be attached to its pendant group at any heteroatom or carbon atom which results in a stable structure and any of the ring atoms may be optionally substituted. Examples of such saturated or partially unsaturated heterocyclic radicals include, without limitation, tetrahydrofuranyl, tetrahydrothiophenyl pyrrolidinyl, piperidinyl, pyrrolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinil, morpholinyl and quinuclidinyl.

[00026] As used herein, the term "partially unsaturated" refers to a portion of the ring that includes at least one double or triple bond. The term "partially unsaturated" is intended to encompass rings having multiple sites of unsaturation, but is not intended to include aryl or heteroaryl moieties as defined herein.

[00027] The term "heteroatom" means one or more of oxygen, sulfur, nitrogen, phosphorus or silicon (including, any oxidized form of nitrogen, sulfur, phosphorus or silicon; the quartenized form of any basic nitrogen or; a replaceable nitrogen of a heterocyclic ring, for example N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl) or NR+ (as in N-substituted pyrrolidinyl)).

[00028] As used herein, the term "pharmaceutically acceptable salt" refers to those salts which are, within the scope of good medical judgment, suitable for use in contact with the tissues of humans and smaller animals without toxicity, irritation, response allergies and the like, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S.M. Berge et al., describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, incorporated herein by reference.

[00029] In certain embodiments, the neutral forms of the compounds are regenerated by contacting the salt with a base or acid and isolating the parent compound in a conventional manner. In some embodiments, the parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents.

[00030] Unless otherwise noted, structures depicted in this document are also intended to include all isomeric (e.g. enantiomeric, diastereomeric, and geometric (or conformational) forms of the structure; e.g. R and S configurations for each asymmetric cent, Z and E double bond isomers, and Z and E conformational isomers. Therefore, single stereochemical isomers, as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of the present compounds are within the scope of the invention. unless otherwise indicated, all tautomeric forms of the compounds of the invention are within the scope of the invention. Additionally, unless otherwise indicated, the structures depicted herein are also intended to include compounds that differ only in the presence of a or more isotopically enriched atoms For example, compounds having the present structures including hydrogen substitution o with deuterium or tritium, or the replacement of a carbon with a carbon enriched with 13C- or 14C are within the scope of this invention. Such compounds are useful, for example, as analytical tools, as probes in biological assays, or as therapeutic agents in accordance with the present invention. compounds

[00031] As described above, in certain embodiments, the present invention provides a compound of formula I:

[00032] or a pharmaceutically acceptable salt thereof, wherein R1 and Ring A are as defined and described herein.

[00033] The compounds of formula I have unexpectedly been found to exhibit advantageous properties over known inhibitors of Btk. In certain embodiments, compounds of formula I have increased potency. Without being bound by any particular theory, the compounds disclosed herein are believed to have improved potency as Btk inhibitors, a better nonspecific profile as measured by hERG inhibition or PXR induction assays, or a combination thereof. . Experimental data showing these advantageous properties are provided in the following Examples.

[00034] In some embodiments, both R1 are hydrogen. In is independently C1-6 aliphatic. In is independently C1-5 aliphatic. In is independently C1-4 aliphatic. In is independently C1-3 aliphatic. In is independently C1-2 aliphatic. In some embodiments, both R1 are methyl.

[00035] In some embodiments, each R1 is independently hydrogen or C1-6 aliphatic. In some embodiments, each R1 is independently hydrogen or C1-5 aliphatic. In some embodiments, each R1 is independently hydrogen or C1-4 aliphatic. In some embodiments, each R1 is independently hydrogen or C1-3 aliphatic. In some embodiments, each R1 is independently hydrogen or C1-2 aliphatic. In some embodiments, each R1 is independently hydrogen or methyl.

[00036] In some embodiments, one R1 is hydrogen and the other R1 is C1-6 aliphatic. In some embodiments, one R1 is hydrogen and the other R1 is methyl. In some embodiments, one R1 is hydrogen and the other R1 is ethyl. In some embodiments, one R1 is hydrogen and the other R1 is C1-6 (cycloalkyl)alkyl. In some embodiments, one R1 is hydrogen and the other R1 is C1-6 (cycloalkyl).

[00037] In some embodiments, one R1 is hydrogen and the other R1 is C1-6aliphatic optionally substituted with -OR, where R is hydrogen or C1-6 aliphatic.

[00038] In some embodiments, one R1 is hydrogen and the other R1 is a heterocyclylalkyl group having 3-7 carbon atoms and 1-3 heteroatoms independently selected from nitrogen, oxygen or sulfur. In some embodiments, one R1 is hydrogen and the other R1 is an optionally substituted 3-7 membered monocyclic heterocycle.

[00039] In some embodiments, two R1 groups are taken together with their intervening atoms to form an optionally substituted saturated or partially unsaturated 3-5 membered monocyclic heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen or sulfur. In some embodiments, two R1 groups are taken together with their intervening atoms to form an optionally substituted piperazine ring.

Em determinadas modalidades, R2 é -Cl. Em outras modalidades, R2 é -F. Em algumas modalidades, R3 é -CF3. Em algumas modalidades, R3 é - OCF3. Em algumas modalidades, R3 é -F.[00040] As described above, Ring A is

In certain embodiments, R2 is -Cl. In other embodiments, R2 is -F. In some embodiments, R3 is -CF3. In some embodiments, R3 is -OCF3. In some embodiments, R3 is -F.

[00041] In certain embodiments, Ring A is selected from the group consisting of:

[00042] In some embodiments, in the compounds described herein, there is a trans stereochemical relationship between the piperidine substituent bearing the carboxamide group and the piperidine substituent bearing the lactam group.

[00043] In some embodiments, the present invention provides a compound of formula II-a:

[00044] or a pharmaceutically acceptable salt thereof, wherein each of R1, R2, and R3 is as defined above and described in classes and subclasses herein.

[00045] In some embodiments, the present invention provides a compound of formula II-b:

[00046] or a pharmaceutically acceptable salt thereof, wherein each of R1, R2, and R3 is as defined above and described in classes and subclasses herein.

[00047] In some embodiments, the present invention provides a compound of formula III:

[00048] or a pharmaceutically acceptable salt thereof, wherein each of R2 and R3 is as defined above and described in classes and subclasses herein.

[00049] In some embodiments, the present invention provides a compound of formula IV:

[00050] or a pharmaceutically acceptable salt thereof, wherein each of R1 and R3 is as defined above and described in classes and subclasses herein. In some embodiments, both R1 are hydrogen. In some embodiments, one R1 is hydrogen and the other R1 is methyl.

[00051] In some embodiments, a compound provided is a compound depicted in Table 1 below, or a pharmaceutically acceptable salt thereof. Table 1 - Selected compounds of formula I.

[00052] The compounds of the invention are synthesized by an appropriate combination of generally known synthetic methods. Techniques useful in synthesizing the compounds of the invention are readily apparent and accessible to those skilled in the relevant art. The discussion below is offered to illustrate some of the various methods available for use in assembling the compounds of the invention. However, the discussion is not intended to define the scope of reactions or reaction sequences that are useful in preparing the compounds of the present invention.

[00053] Compounds of formula I can be prepared generally according to Scheme 1. Scheme 1

[00054] Compounds of formula I can also be prepared generally according to Schemes 2, 3, 3a, 4, 4a, 5, or 5a.

[00055] The PG, PG1, PG2, and PG3 groups of the compounds in Schemes 1 through 5a are each independently a suitable protecting group. Such ester and amine protecting groups are known in the art and are described in detail in Protecting Groups in Organic Synthesis, T.W. Greene and P.G.M. Wuts, 3rd edition, John Wiley & Sons, 1999, the entirety of which is incorporated herein by reference. In some embodiments, a protecting group is a Boc group.

[00056] In certain embodiments, each of the synthetic steps in Schemes 1 through 5a may be performed sequentially with isolation of each intermediate performed after each step. Alternatively, each of the steps as described in Schemes 1, 2, 3 and 4 above may be carried out in a manner whereby no isolation of one or more intermediates is carried out.

[00057] In certain embodiments, all steps of the aforementioned synthesis can be performed to prepare the desired end product. In other embodiments, two, three, four, five or more sequential steps may be performed to prepare an intermediate or desired end product. Uses, Formulation and Administration

[00058] In certain embodiments, the compounds of the present invention are for use in medicine. In some embodiments, the present invention provides a method of decreasing the enzymatic activity of a kinase in the Tec kinase family (e.g., Tec, Btk, Itk, Txk, Lck and Bmx). In some embodiments, these methods include contacting a Tec kinase family kinase with an effective amount of a Tec kinase family inhibitor. Therefore, the present invention further provides methods of inhibiting Tec kinase family enzyme activity by contacting a Tec kinase family member with a Tec kinase family inhibitor of the present invention. As used herein, the term "member of the Tec kinase family" refers to any non-receptor tyrosine kinase in the Tec kinase family. In some embodiments, members of the Tec kinase family are Tec, Btk, Itk, Txk, Lck, and Bmx.

[00059] In some embodiments, the present invention provides methods of decreasing the enzymatic activity of Btk. In some embodiments, these methods include contacting the Btk with an effective amount of a Btk inhibitor. Therefore, the present invention further provides methods of inhibiting Btk enzymatic activity by contacting a Btk with a Btk inhibitor of the present invention.

[00060] Btk enzymatic activity as used in this document refers to the enzymatic activity of Btk kinase. For example, where Btk enzymatic activity is decreased, PIP3 binding and/or PLCY phosphorylation is decreased. In some embodiments, the half maximal inhibitory concentration (IC50) of the Btk inhibitor against Btk is less than 1 μM. In some embodiments, the IC50 of the Btk inhibitor against Btk is less than 500 nM. In some embodiments, the IC50 of the Btk inhibitor against Btk is less than 100 nM. In some embodiments, the IC50 of the Btk inhibitor against Btk is less than 10 nM. In some embodiments, the IC50 of the Btk inhibitor against Btk is less than 1 nM. In some embodiments, the IC50 of the Btk inhibitor against Btk is 0.1 nM to 10 μM. In some embodiments, the IC50 of the Btk inhibitor against Btk is 0.1 nM to 1 μM. In some embodiments, the IC50 of the Btk inhibitor against Btk is from 0.1 nM to 100 nM. In some embodiments, the IC50 of the Btk inhibitor against Btk is from 0.1 nM to 10 nM.

[00061] In some embodiments, inhibitors of these Tec kinases are useful for treating diseases and disorders that can be ameliorated by inhibiting (ie, decreasing) the enzymatic activity of one or more Tec kinases. The compounds of the invention are effective inhibitors of Tec family kinases and thus would be useful in the treatment of diseases associated with the activity of one or more of the Tec family kinases. The term "diseases" means diseases, syndromes or disease symptoms. Thus, the present invention provides methods of treating autoimmune diseases, inflammatory diseases and cancers in a subject in need. Such methods include administering to the subject a therapeutically effective amount of a Tec, Btk, Itk, Txk, Lck, and/or Bmx kinase inhibitor.

[00062] The term "autoimmune disorders" includes diseases or disorders that involve an inappropriate immune response against native antigens, such as acute disseminated encephalomyelitis (ADEM), Addison's disease, alopecia areata, antiphospholipid antibody syndrome (APS), hemolytic anemia, autoimmune hepatitis, bullous pemphigoid (BP), celiac disease, dermatomyositis, type 1 diabetes mellitus, Goodpasture syndrome, Graves disease, Guillain-Barré syndrome (GBS), Hashimoto's disease, idiopathic thrombocytopenic purpura, lupus or systemic lupus erythematosus ( SLE), mixed connective tissue disease, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, hemophilia with inhibitors, pernicious anemia, polymyositis, primary biliary cirrhosis, Sjogren's syndrome, temporal arteritis, and Wegener's granulomatosis. The term "inflammatory diseases" includes diseases or disorders involving acute or chronic inflammation, such as allergies, asthma (e.g. allergic asthma), atopic dermatitis, prostatitis, glomerulonephritis, pelvic inflammatory disease (PID), inflammatory bowel disease (IBD, for example, Crohn's disease, ulcerative colitis), reperfusion injury, rheumatoid arthritis, transplant rejection (including transplant patients with a positive match), and vasculitis. In certain embodiments, the present invention provides methods of treating diseases, disorders or conditions that have been approved for treatment with rituximab (a monoclonal antibody against CD20), including non-Hodgkin's lymphoma (NHL), chronic lymphocytic leukemia (CLL), RA , Wegener's granulomatosis (WG), and microscopic polyangiitis (MPA). In some embodiments, the present invention provides a method of treating rheumatoid arthritis (RA), SLE or atopic dermatitis using the compounds disclosed herein.

[00063] The term "cancer" includes diseases or disorders involving abnormal cell growth and/or proliferation, such as glioma, thyroid carcinoma, breast carcinoma, lung cancer (e.g., small cell lung carcinoma, non-small cell lung), gastric carcinoma, gastrointestinal stromal tumors, pancreatic carcinoma, bile duct carcinoma, ovarian carcinoma, endometrial carcinoma, prostate carcinoma, renal cell carcinoma, lymphoma (e.g. anaplastic large cell lymphoma), leukemia (eg, acute myeloid leukemia, T-cell leukemia, chronic lymphocytic leukemia), multiple myeloma, malignant mesothelioma, malignant melanoma, and colon cancer (eg, high microsatellite instability colorectal cancer). In some embodiments, the present invention provides a method of treating leukemia or lymphoma.

[00064] The term "subject" as used herein refers to a mammal to which a pharmaceutical composition is administered. Exemplary subjects include humans, as well as veterinary and laboratory animals, such as horses, pigs, cattle, dogs, cats, rabbits, rats, mice, and aquatic mammals. Selected Indications and Inhibition of B Cells

[00065] As described above, the compounds provided are useful for the treatment of disease, including RA and SLE. As described in more detail below, these diseases are affiliated with B cells. Thus, the present disclosure encompasses recognition that the compounds provided are useful as therapeutics for these and other indications.

[00066] Dysregulation of the immune system is central to the pathogenesis (Panayi GS, et al. Rheum Dis Clin North Am 2001; 27:317-334) of RA. Although most of the leukocytes infiltrating the synovium are T lymphocytes (primarily activated CD4+ T cells) and cells of monocytic/macrophage origin (which release proinflammatory cytokines such as IL-1, TNF-alpha and IL-6 and proteolytic enzymes). including collagenases and metalloproteinases), B cells and plasma cells are also found in synovial fluid (Zhang Z, Bridges SL. Rheum Dis Clin North Am 2001; 27:335-353). A clear role for B cells and their associated effector functions in RA has been demonstrated by the efficacy of rituximab, a therapeutic for selective B cell depletion, which is approved for the treatment of RA (Cohen SB, et al.; REFLEX Trial Group Arthritis Rheum, September 2006; 54(9):2793-806 ).

[00067] Although the etiology of SLE is not fully understood, pathogenic autoantibodies and the deposition of immune complexes are felt to be critical for the development of widespread tissue damage (Klippel JH, et al. Primer on the rheumatic diseases. Atlanta: Arthritis Foundation ; 2001). Autoantibody and immune complex-mediated activation can be studied by measuring the inhibition of macrophage activation by macrophages stimulated through Fc receptors (see the exemplification - FCYR activation of primary human macrophages). Loss of tolerance to autoantigens ultimately led to stimulation of B cells to produce autoantibodies generally directed against nuclear or cytoplasmic components. Antibodies against nuclear components (antinuclear antibodies) target nuclear antigens including DNA (typical double-stranded DNA [dsDNA]), RNA, histones, and small nuclear ribonucleoproteins. These antibodies combine with self-antigens to form immune complexes that are deposited in tissues, stimulate inflammatory reactions, and lead to tissue damage. In addition to their roles in pathogenic autoantibody production, B cells also function as antigen-presenting cells (APCs) for T cells, thus playing a role in initiating an antigen-specific response. Given the central role of the humoral branch of the immune system in the pathogenesis of SLE, B cells or the B cell pathway represent desirable therapeutic targets. Belimumab, a newly approved monoclonal antibody for SLE, blocks the binding of BAFF to its receptors which are expressed B cells. These receptors serve to activate and potentiate B cell survival consistent with the reduction in circulating B cells seen after belimumab treatment. See also Chan OT, et al. Immunol Rev. 1999b;169:107-121; Navarra SV, et al. Lancet. Feb 26, 2011;377(9767):721-31; Furie R, et al. Arthritis Rheum. December, 2011;63(12):3918-30. The role of B cells and myeloid lineage cells in autoimmune diseases such as SLE is further supported by a recent publication describing the efficacy in an animal model of preclinical SLE when mice are treated with an irreversible Btk inhibitor of small molecule (Honigberg, L.A. PNAS. 2010; 107: 13075). combinations

[00068] In certain embodiments, a compound of the present invention is administered in combination with another agent. In some embodiments, a compound of the present invention is useful for the treatment of RA and is administered in combination with disease-modifying antirheumatic drugs (DMARDs), including, without limitation: methotrexate, abatacept, azathioprine, certolizumab, chloroquine and hydroxychloroquine, cyclosporine, D-penicillamine, adalimumab, etanercept, golimumab, gold salts (including auranofin and sodium aurothiomalate), infliximab, leflunomide, minocycline, rituximab, sulfasalazine, tocilizumab, or combinations thereof. In some embodiments, a compound of the present invention is administered in combination with an NSAID or corticosteroid. In some embodiments, a compound of the present invention is useful for the treatment of SLE and is administered in combination with an agent for the treatment of SLE, including, without limitation: corticosteroids, antimalarials, belimumab, mycophenolate mofetil (MMF) or mycophenolate mofetil. sodium, azathioprine, or combinations thereof. In some embodiments, a compound of the present invention is useful for the treatment of atopic dermatitis and is administered in combination with a topical agent for the treatment of atopic dermatitis, including, without limitation: topical steroids, tacrolimus, methotrexate, mometasone furoate (MMF ), azathioprine, retinoids, or combinations thereof. Essay

[00069] To develop useful Tec kinase family inhibitors, candidate inhibitors capable of decreasing the enzymatic activity of the Tec kinase family can be identified in vitro. The activity of the inhibitor compounds can be analyzed using methods known in the art and/or those methods disclosed herein.

[00070] Compounds that decrease the enzymatic activity of Tec kinase family members can be identified and tested using a biologically active Tec kinase family member, either recombinantly or naturally occurring. Tec kinases can be found in native cells, isolated in vitro, or co-expressed or expressed in a cell. The measurement of the reduction in the enzymatic activity of the Tec kinase family member in the presence of an inhibitor relative to the activity in the absence of the inhibitor can be performed using a variety of methods known in the art, such as the POLYGAT-LS assays described below in the Examples. . Other methods for analyzing the activity of Btk and other Tec kinases are known in the art. The selection of appropriate test methods is within the skill of those skilled in the art.

[00071] Once the compounds are identified as capable of reducing the enzymatic activity of members of the Tec kinase family, the compounds can be further tested for their ability to selectively inhibit a member of the Tec kinase family relative to other enzymes.

[00072] The compounds can be further tested in cell models or animal models for their ability to cause detectable changes in phenotype related to an activity of the Tec kinase family member. In addition to cell cultures, animal models can be used to test inhibitors of the Tec kinase family member for its ability to treat autoimmune diseases, inflammatory diseases or cancer in an animal model. Pharmaceutical Compositions

[00073] In another aspect, the present invention provides pharmaceutical compositions comprising a compound of formula I or a compound of formula I in combination with a pharmaceutically acceptable excipient (e.g., carrier).

[00074] Pharmaceutical compositions include optical isomers, diastereomers or pharmaceutically acceptable salts of the inhibitors disclosed herein. The compound of formula I included in the pharmaceutical composition may be covalently linked to a carrier moiety as described above. Alternatively, the compound of formula I included in the pharmaceutical composition is not covalently linked to a carrier moiety.

[00075] A "pharmaceutically acceptable carrier", as used herein, refers to pharmaceutical excipients, for example, pharmaceutically, physiologically acceptable, organic or inorganic carrier substances suitable for enteral and parenteral application that do not deleteriously react with the agent. active. Suitable pharmaceutically acceptable carriers include water, salt solutions (such as Ringer's solution), alcohols, oils, gelatins and carbohydrates such as lactose, amylose or starch, fatty acid esters, hydroxymethyl cellulose and polyvinyl pyrrolidine. Such preparations can be sterilized and, if desired, mixed with auxiliary agents, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts to influence the osmotic pressure, buffers, coloring and/or aromatic substances and the like, which do not react in any way. deleterious form with the compounds of the invention.

[00076] The compounds of the invention may be administered alone or may be co-administered to the subject. Co-administration is intended to include the simultaneous or sequential administration of the compounds individually or in combination (more than one compound). The preparations can also be combined, when desired, with other active substances (eg to reduce metabolic degradation).

[00077] The compounds of the present invention can be prepared and administered in a wide variety of oral, parenteral and topical dosage forms. Thus, compounds of the present invention may be administered by injection (e.g., intravenously, intramuscularly, intracutaneously, subcutaneously, intraduodenally, or intraperitoneally). In addition, the compounds described herein can be administered by inhalation, for example, intranasally. Additionally, compounds of the present invention can be administered transdermally. It is also anticipated that various routes of administration (e.g., intramuscular, oral, transdermal) may be used to administer the compounds of the invention.

[00078] For the preparation of pharmaceutical compositions from the compounds of the present invention, pharmaceutically acceptable carriers can be solid or liquid. Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories and dispersible granules. A solid carrier can be one or more substances that can also act as diluents, flavoring agents, binders, preservatives, tablet disintegrating agents, or an encapsulating material.

[00079] In powders, the carrier is a finely divided solid in a mixture with the finely divided active component. In tablets, the active component is mixed with the carrier having the necessary binding properties in suitable proportions and compacted into the desired shape and size.

[00080] Powders and tablets preferably contain from 5% to 70% of the active compound. Suitable carriers are magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, a low-melting wax, cocoa butter, and the like. The term "preparation" is intended to include formulating the active compound with an encapsulating material as a carrier providing a capsule in which the active component, with or without other carriers, is surrounded by a carrier, which is therefore in association with he. Similarly, cachets and inserts are included. Tablets, powders, capsules, pills, cachets and lozenges can be used as solid dosage forms suitable for oral administration.

[00081] For the preparation of suppositories, a low-melting wax, such as a mixture of fatty acid glycerides or cocoa butter, is first melted and the active component is homogeneously dispersed therein, as by stirring. The molten homogeneous mixture is then poured into suitable sized molds, allowed to cool and thereby solidify.

[00082] Liquid form preparations include solutions, suspensions and emulsions, for example water or water/propylene glycol solutions. For parenteral injection, liquid preparations may be formulated in solution in aqueous polyethylene glycol solution.

[00083] When parenteral application is necessary or desired, particularly suitable mixtures for the compounds of the invention are injectable, sterile solutions, preferably oily or aqueous solutions, as well as suspensions, emulsions or implants, including suppositories. In particular, carriers for parenteral administration include aqueous solutions of dextrose, saline, pure water, ethanol, glycerol, propylene glycol, peanut oil, sesame oil, polyoxyethylene block polymers, and the like. Ampoules are convenient unit dosages. Compounds of the invention may also be incorporated into liposomes or administered via pumps or transdermal patches. Pharmaceutical mixtures suitable for use in the present invention include those described, for example, in Pharmaceutical Sciences (17th Ed., Mack Pub. Co., Easton, PA) and WO 96/05309, the teachings of both of which are hereby incorporated herein. of this by reference.

[00084] Aqueous solutions suitable for oral use can be prepared by dissolving the active component in water and adding suitable coloring, flavoring, stabilizing and thickening agents as desired. Aqueous suspensions suitable for oral use can be made by dispersing the finely divided active component in water with viscous material, such as natural or synthetic gums, resins, methyl cellulose, sodium carboxymethyl cellulose, and other known suspending agents.

[00085] Also included are solid form preparations which are intended to be converted, immediately before use, to liquid form preparations for oral administration. These liquid forms include solutions, suspensions and emulsions. These preparations may contain, in addition to the active component, coloring, flavorings, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizing agents, and the like.

[00086] The pharmaceutical preparation is preferably in unit dosage form. In this form of preparation it is subdivided into unit doses containing appropriate amounts of the active component. The unit dosage form can be a packaged preparation, the package containing discrete amounts of the preparation, such as tablets, capsules, and powders in packaged vials or ampoules. Furthermore, the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form.

[00087] The amount of active ingredient in a unit dose preparation can be varied or adjusted from 0.1mg to 10000mg, more normally 1.0mg to 1000mg, more normally 10mg to 500mg, according to the application specificity and the potency of the active component. The composition may, if desired, also contain other compatible therapeutic agents.

[00088] Some compounds may have limited solubility in water and therefore may require a surfactant or other appropriate co-solvent in the composition. Such co-solvents include: Polysorbate 20, 60 and 80; Pluronic F-68, F-84 and P-103; cyclodextrin; and polyoxyl 35 castor oil. These co-solvents are normally employed at a level between about 0.01% and about 2% by weight.

[00089] Viscosity greater than that of simple aqueous solutions may be desirable to decrease variability in the distribution of formulations, to decrease physical separation of components of a suspension or emulsion from the formulation, and/or to otherwise improve the formulation . Such viscosity building agents include, for example, polyvinyl alcohol, polyvinyl pyrrolidone, methyl cellulose, hydroxypropyl methyl cellulose, hydroxyethyl cellulose, carboxymethyl cellulose, hydroxypropyl cellulose, chondroitin sulfate and salts thereof, hyaluronic acid and salts thereof, and combinations of the foregoing. Such agents are normally employed at a level between about 0.01% and about 2% by weight.

[00090] The compositions of the present invention may additionally include components to provide sustained release and/or comfort. These components include high molecular weight anionic mucomimetic polymers, gelling polysaccharides, and finely divided drug carrier substrates. These components are discussed in more detail in Pat. U.S. No. 4,911,920; 5,403,841; 5,212,162; and 4,861,760. All contents of these patents are incorporated herein in their entirety by reference for all purposes. Effective Doses

[00091] The pharmaceutical compositions provided by the present invention include compositions, wherein the active ingredient is contained in a therapeutically effective amount, i.e., in an amount effective to achieve its intended purpose. The actual amount effective for a specific application will depend, inter alia, on the condition being treated. For example, when administered in methods for treating cancer, such compositions will contain an amount of active ingredient effective to achieve the desired result (e.g., decrease in the number of cancer cells in a subject).

[00092] The dosage and frequency (single or multiple dose) of the administered compound may vary depending on a variety of factors, including the route of administration; recipient's size, age, sex, health, body weight, body mass index, and diet; nature and degree of symptoms of the disease being treated (eg, the disease responsive to Btk inhibition); presence of other diseases or other health-related problems; type of simultaneous treatment; and complications of any disease or treatment regimen. Other therapeutic regimens or agents can be used in conjunction with the methods and compounds of the invention.

[00093] For any compound described herein, the therapeutically effective amount can be initially determined from cell culture assays. Target concentrations will be those concentrations of the active compound(s) that are capable of decreasing the kinase enzymatic activity as measured, for example, using the methods described.

[00094] Therapeutically effective amounts for use in humans can be determined from animal models. For example, a dose for humans can be formulated to achieve a concentration that has been found to be effective in animals. Dosage in humans can be adjusted by monitoring kinase inhibition and adjusting the dosage up or down as described above. In certain embodiments, the dose administered is in the range of about 10 mg to about 1000 mg per day, either once, twice, or more than twice a day.

[00095] Dosages can be varied depending on the requirements of the patient and the compound being employed. The dose administered to a patient, in the context of the present invention, should be sufficient to effect a beneficial therapeutic response in the patient over time. Dose size will also be determined by the existence, nature and degree of any adverse side effects. Treatment is usually started with smaller dosages, which are less than the optimal dose of the compound. Thereafter, the dosage is increased in small increments until the optimal effect under the circumstances is reached. In some embodiments, the dosage range is 0.001% to 10% w/v. In some embodiments, the dosage range is 0.1% to 5% w/v.

[00096] Dosage amounts and intervals can be individually adjusted to provide effective levels of administered compound for the specific clinical indication being treated. This will provide a therapeutic regimen that is commensurate with the severity of the individual's disease state.

[00097] In order that the invention described in this document may be more fully understood, the following examples are represented. It is to be understood that these examples are for illustrative purposes only and should not be construed as limiting this invention in any way. Exemplification

[00098] As depicted in the Examples below, in certain exemplary embodiments, compounds are prepared according to the following general procedures. It will be appreciated that although the general methods depict the synthesis of certain compounds of the present invention, the following general methods, and other methods known to one of skill in the art, may be applied to all compounds and subclasses and species of each such compound, as described in this document. Example 1 Synthesis of (3'R,4'S)-1'-tert-butyl 4'-ethyl 2-oxo-[1,3'-bipiperidine]-1',4'-dicarboxylate

[00099] Preparation of ethyl 3-oxopiperidine-4-carboxylate intermediate. Ethyl 1-Benzyl-3-oxopiperidine-4-carboxylate 1 (15.0 g, 50.5 mmol, 1.0 equiv) was hydrogenated in the presence of 10% Pd/C catalyst (1.5 g) under H2 at atmospheric pressure in MeOH (250 mL) for 16 hours. The catalyst was filtered and the solvent was concentrated in vacuo to give ethyl 3-oxopiperidine-4-carboxylate 2 as a pale yellow solid (10.2 g, yield: 98.0%). ESI-MS (M+H) +: 172.1. 1H NMR (400 MHz, DMSO-d6) δ: 4.23 (q, 2H), 3.75 (s, 2H), 3.37 (s, 2H), 3.20-3.16 (m, 2H ), 2.44 (t, 1H), 1.25 (t, 3H).

[000100] Preparation of 1-tert-butyl 4-ethyl 3-oxopiperidine-1,4-dicarboxylate. Ethyl 3-Oxopiperidine-4-carboxylate 2 (10.2 g, 60.0 mmol, 1.0 equiv) was dissolved in dry MeOH (200 mL), and Et 3 N (33.1 mL, 240 mmol, 4.0 equiv) has been added. The mixture was stirred for one hour and Boc2O (19.5 g, 90.0 mmol, 1.5 equiv) was added and stirred for 16 hours. The solvent was concentrated in vacuo and the crude product was purified by column chromatography (silica, petroleum ether/EtOAc =9:1) to give a light yellow oil of 1-tert-3-oxopiperidine-1,4-dicarboxylate. butyl 4-ethyl 3 (11.5 g, yield: 86%). ESI-MS (M+H-56) +: 216.0. 1H NMR (400 MHz, CDCl3) δ: 4.24 (q, 2H), 4.03 (s, 2H), 3.49 (t, 2H), 2.33 (t, 2H), 1.47 ( s, 9H), 1.31 (t, 3H).

[000101] Preparation of (S)-1-tert-butyl 4-ethyl 3-((1-phenylethyl)amino)-5,6-dihydropyridine-1,4-(2H)-dicarboxylate. In a dry flask equipped with a Dean-Stark trap and reflux condenser, 1-tert-butyl 4-ethyl 3-oxopiperidine-1,4-dicarboxylate 3 (10.0 g, 37.0 mmol, 1 .1 equiv) was dissolved in toluene (100 mL). S-(-)-α-Methylbenzylamine (4.9 g, 40.5 mmol, 1.1 equiv) and p-toluenesulfonic acid monohydrate (0.7 g, 3.7 mmol, 0.1 equiv) were added and the mixture was heated to reflux for 16 hours. The crude reaction mixture was concentrated in vacuo to give (S)-1-tert-butyl 4-3-((1-phenylethyl)amino)-5,6-dihydropyridine-1,4(2H)-dicarboxylate ethyl 4 (12.0 g, Y: 88%) as a thick orange oil which was used in the next step without further purification, ESI-MS (M+H) +: 375.2.

[000102] Preparation of 1-tert-Butyl 4-ethyl 3-(((S)1-phenylethyl)amino)-5,6-dihydropyridine-1,4(2H)-dicarboxylate. 1-tert-Butyl 4-ethyl 3-(((S)-1-phenylethyl)amino)piperidine-1,4-dicarboxylate 4 (11.2 g, 30.0 mmol, 1.0 equiv) was dissolved in a mixture of CH3CN (60 mL) and acetic acid (60 mL) and cooled to 0°C. NaBH(OAc)3 (19.0 g, 90.0 mmol, 3.0 equiv) was slowly added and the reaction mixture was allowed to stir for two hours at 0°C. Saturated NaHCO3 was slowly added to neutralize the solution to keep the internal temperature of the flask below 10°C. The mixture was extracted with EtOAc (50 mL x 3). The combined organic layer was dried (Na2SO4), filtered, concentrated in vacuo, and then purified by column chromatography (silica, petroleum ether/EtOAc =9:1) to give 3-(((S)1-phenylethyl)amino 4-ethyl)-5,6-dihydropyridine-1,4(2H)-dicarboxylate 5 (8.2 g, Y: 73%) as light yellow oil. ESI-MS (M+H) +: 377.2. 1H NMR (400 MHz, CD3OD) δ: 7.31-7.22 (m, 5H), 4.20 (q, 2H), 4.11-3.86 (m, 3H), 3.15 (s , 1H), 3.00-2.90 (m, 2H), 2.64 (d, 2H), 1.87-1.85 (m, 1H), 1.68 (s, 1H), 1. 50-1.25 (m, 15H).

[000103] Preparation of trans-1-tert-butyl 4-ethyl 3-(((S)-1-phenylethyl)amino)piperidine-1,4-dicarboxylate. 1-tert-Butyl 4-ethyl 3-(((S)-1-phenylethyl)amino)piperidine-1,4-dicarboxylate 5 (8.0 g, 21.2 mmol, 1.0 equiv) was dissolved in dry EtOH (20 mL) under N2. Into a separate flame-dried Schlenk flask was placed dry EtOH (150 mL), and sodium (0.450 g, 63.6 mmol, 3.0 equiv) was added portionwise under N2. The mixture was kept under N2 and vented to remove emitted gases until all the sodium had dissolved. The clear solution of 1-tert-butyl 4-ethyl 3-(((S)-1-phenylethyl)amino)piperidine-1,4-dicarboxylate was then transferred to the NaOEt solution, and the mixture was stirred at 80° C under N2 for 16 hours. The solvent was removed in vacuo, and brine (150 mL) was added and the mixture was brought to pH =10 with 1N NaOH and extracted with EtOAc (100 mL x 3). The combined organic layers were dried (Na2SO4) and concentrated in vacuo. The residue was purified by column chromatography (silica, petroleum ether/EtOAc = 5:1) to give (trans)-1 3-(((S)-1-phenylethyl)amino)piperidine-1,4-dicarboxylate -tert-butyl 4-ethyl 6 as a slightly yellow solid (3.7 g, yield: 46%). ESIMS (M+H) +: 377.2.

[000104] Preparation of trans-1-tert-butyl 4-ethyl 3-aminopiperidine-1,4-dicarboxylate. trans-1-tert-butyl 4-ethyl 3-(((S)-1-phenylethyl)amino)piperidine-1,4-dicarboxylate 6 (3.7 g, 8.3 mmol, 1.0 equiv) was hydrogenated in the presence of 10% Pd/C catalyst (0.37 g) under H 2 at atmospheric pressure 30 in MeOH (100 mL) at 50°C for 8 h. The catalyst was filtered and the solvent was removed in vacuo to give (trans)-1-tert-butyl 4-ethyl 3-aminopiperidine-1,4-dicarboxylate 7 as a pale yellow oil (2.5 g, yield: 92 %). ESI-MS (M+H) +: 273.1. 1H NMR (400 MHz, CDCl 3 ) δ: 4.18 (q, 2H), 3.97-3.94 (m, 2H), 3.37 (s, 1H), 3.07-3.02 (m , 1H), 2.89-2.85 (m, 1H), 2.60-2.55 (m, 1H), 2.01-1.91 (m, 1H), 1.70-1.54 (m, 3H), 1.46 (s, 9H), 1.28 (t, 3H).

[000105] Synthesis of trans-1-tert-butyl 4-ethyl 3-(5-bromopentanamido)piperidine-1,4-dicarboxylate. To a solution of trans-1-tert-butyl 4-ethyl 3-aminopiperidine-1,4-dicarboxylate 7 (2.5 g, 9.2 mmol, 1.0 equiv) in CH2Cl2 (50 mL), Et3N ( 2.5 mL, 18.4 mmol, 2.0 equiv) was added at room temperature. After the reaction solution was stirred at room temperature for 10 min, 5-bromovaleryl chloride (1.9 g, 9.6 mmol, 1.05 eq) was added. The reaction solution was stirred at room temperature for two hours. The mixture was quenched with H2O (20 mL) and extracted with CH2Cl2 (50 mL x 3). The organic layer was collected, concentrated in vacuo, and the residue was purified by column chromatography (silica, petroleum ether/EtOAc =1:1) to give 3-(5-bromopentanamido)piperidine-1,4-dicarboxylate ( trans)-1-tert-butyl 4-ethyl 8 as a yellow oil (3.2 g, yield: 80%). ESIMS (M+H-56) +: 379.0. 1H NMR (400 MHz, CDCl 3 ) δ: 5.99 (d, 1H), 4.394.38 (m, 1H), 4.15 (q, 2H), 3.79-3.74 (m, 1H), 3.66-3.60 (m, 1H), 3.41 (t, 2H), 3.30-3.26 (m, 1H), 3.21-3.14 (m, 1H), 2, 78-2.74 (m, 1H), 2.19 (t, 2H), 1.99-1.85 (m, 3H), 1.80-1.72 (m, 3H), 1.45 ( s, 9H), 1.27 (t, 3H). Synthesis of trans-1'-tert-butyl 4'-ethyl 2-oxo-[1,3'-bipiperidine]-1',4'-dicarboxylate. To a solution of trans-1-tert-butyl 4-ethyl 3-(5-bromopentanamido)piperidine-1,4-dicarboxylate 8 (3.0 g, 6.9 mmol, 1.0 equiv) in THF (20 mL), NaH (276 mg, 6.9 mmol, 1.0 equiv) was added carefully in small portions at 0°C. The reaction solution was stirred under reflux for 4 hours. The mixture was quenched with H 2 O (20 mL), and extracted with EtOAc (30 mL x 3). The organic layer was collected, dried (Na2SO4), filtered and concentrated in vacuo. The residue was purified by column chromatography (silica, petroleum ether/EtOAc = 1:2) to give (trans)-1'2-oxo-[1,3'-bipiperidine]-1',4'-dicarboxylate -tert-butyl 4'-ethyl 9 as a slightly yellow oil (2.1 g, yield: 88%). ESI-MS (M+H-56)+: 299.1. 1H NMR (400 MHz, CDCl3) δ: 4.10 (q, 4H), 3.38-3.19 (m, 4H), 2.70-2.61 (m, 1H), 2.36-2 .31 (m, 2H), 1.95-1.92 (m, 1H), 1.75-1.71 (m, 6H), 1.46 (s, 9H), 1.23 (t, 3H ). Example 2 Preparation of trans-1'-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-2-oxo-[1,3'-bipiperidine]-4'-carboxamide

[000106] Synthesis of trans-1'-tert-butyl-4'-ethyl-3-iodo-2-oxo-[1,3'-bipiperidine]-1',4'-dicarboxylate. To a solution of trans-1'-tert-butyl 4'-ethyl 2-oxo-[1,3'-bipiperidine]-1',4'-dicarboxylate 8 (141 mg, 2.58 mmol, 1.0 equiv ) in dry toluene (10 mL) at 0°C, TMEDA (0.89 g, 7.7 mmol, 3.0 equiv) and TMSCl (0.6 mg, 1.0 mmol, 2.0 equiv) were added successively under N2. After 0.5 h, I2 (0.98 g, 3.87 mmol, 1.5 equiv) was carefully added in small portions. The reaction solution was stirred at 0°C at room temperature for 16 hours. The mixture was diluted with EtOAc (100 mL), washed with saturated Na2S2O3 (20 mL x 2) and brine (20 mL), dried (Na2SO4), filtered and concentrated in vacuo. Crude product 9 (2.2 g, Y: 81%) was used directly in the next step without further purification. ESI-MS (M+H-56)+: 424.9.1H NMR (400 MHz, CDCl 3 ) δ: 4.78-4.73 (m, 1H), 4.19-4.04 (m, 4H) , 3.55-3.30 (m, 4H), 3.243.16 (m, 2H), 2.73-2.60 (m, 1H), 2.22-2.14 (m, 2H), 1 .96-1.78 (m, 2H), 1.70-1.60 (m, 1H), 1.44 (s, 9H), 1.25 (t, J = 7.2 Hz, 3H).

[000107] Synthesis of trans-1'-tert 3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-2-oxo-[1,3'-bipiperidine]-1',4'-dicarboxylate -butyl 4'-ethyl. A 1.0 M solution of lithium bis(trimethyldisyl)amide in THF (13 mL, 12 mmol, 2.0 equiv) was added via an addition funnel at 10-15°C to a solution of 3-chloro- 5-(Trifluoromethyl)aniline (15 g, 78 mmol, 1.2 equiv) in THF (13 mL). The mixture was allowed to stir at room temperature for 20 minutes and a solution of trans-1'-tert-butyl-4'-ethyl-3-iodo-2-oxo-[1,3'-bipiperidine]-1',4 Crude '-dicarboxylate 9 (3.7 g, 65 mmol, 1.0 equiv) in THF (13 mL) was added via an addition funnel at 10-15°C over 30 minutes. After the addition, the reaction was allowed to stir at room temperature for 30 minutes. Upon completion, the reaction was cooled to 5°C and quenched slowly with water (10 mL), keeping the temperature below 20°C. The quenched reaction was extracted with EtOAc (2 x 30 mL). The combined organic layers were washed with saturated brine (30 mL), dried (Na 2 SO 4 ), filtered and concentrated in vacuo. The resulting crude product was purified with silica gel eluting with a gradient of 10% to 75% EtOAc in heptanes to give the desired product 10. ESI-MS (M+H-56)+: 463.1. 1H NMR (400 MHz, CDCl 3 ) δ: 6.92 (s, 1H), 6.71-6.69 (m, 2H), 4.17-4.06 (m, 4H), 3.78-3 .68 (m, 2H), 3.46-3.36 (m, 3H), 3.23-3.07 (m, 2H), 2.73-2.65 (m, 1H), 2.44 -2.37 (m, 1H), 2.03-1.85 (m, 3H), 1.71-1.61 (m, 2H), 1.46 (s, 9H), 1.27-1 .19 (m, 3H).

[000108] Synthesis of trans-1'-(tert-butoxycarbonyl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-2-oxo-[1,3'-bipiperidine]-4' acid -carboxylic. To a solution of trans-1'-tert-1',4'-dicarboxylate 3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-2-oxo-[1,3'-bipiperidine]-1',4'-dicarboxylate 4'-ethylbutyl 10 (180 mg, 0.33 mmol, 1.0 equiv) in EtOH (5 mL) was added NaOH (40 mg, 0.99 mmol, 3.0 equiv) and the solution was stirred at 80°C. °C for one hour. The solvent was concentrated in vacuo and the residue suspended in water (10ml) and adjusted to pH=6 with HCl (4N). The precipitate was filtered to give (trans)-1'-(tert-butoxycarbonyl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-2-oxo-[1,3'-bipiperidine acid ]-4'-carboxylic acid 11 (150 mg, Y: 82%) as a yellow solid which was used in the next step without further purification. ESI-MS (M+H-85) +: 463.1. 1H NMR (400 MHz, CDCl3) δ: 6.85 (s, 1H), 6.82 (s, 1H), 6.78 (s, 1H), 4.12-3.96 (m, 4H), 3.53-3.37 (m, 2H), 3.11-3.04 (m, 2H), 2.75-2.67 (m, 1H), 2.24-2.18 (m, 1H ), 1.98-1.89 (m, 3H), 1.71-1.58 (m, 2H), 1.44 (s, 9H). Synthesis of trans-tert-butyl 4'-carbamoyl-3-((3-chloro-5-(trifluoromethyl)phenyl)--amino)-2-oxo-[1,3'-bipiperidine]-1'-carboxylate . To trans 1'-(tert-butoxycarbonyl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-2-oxo-[1,3'-bipiperidine]-4'-carboxylic acid solution 11 (70 mg, 0.14 mmol, 1.0 equiv) in DMF (2 mL), NH4Cl (22 mg, 0.41 mmol, 3.0 equiv) was added, HBTU (103 mg, 0.270 mmol, 2.0 equiv) and DIPEA (52 mg, 0.41 mmol, 3.0 equiv). The reaction solution was stirred at room temperature for 16 hours, diluted with EtOAc (10 mL) and washed with water (5 mL) and brine (5 mL). The organic phase was separated and concentrated in vacuo to give a crude oil which was purified by pre-HPLC (MeOH/H 2 O with 0.05% TFA as mobile phase) to give the compound 4'-carbamoyl-3-((3- (trans)-tert-butyl 12 chloro-5-(trifluoromethyl)phenyl)amino)-2-oxo-[1,3'-bipiperidine]-1'-carboxylate (60 mg, yield: 86%) as a solid clear. ESI-MS (M+H-56)+: 463.1. 1H NMR (400 MHz, CD3OD) δ: 6.87-6.86 (m, 1H), 6.846.83 (m, 1H), 6.80 (s, 1H), 4.11-4.03 (m , 3H), 3.53-3.35 (m, 2H), 3.20-3.08 (m, 2H), 2.77-2.74 (m, 1H), 2.25-2.18 (m, 1H), 1.99-1.88 (m, 3H), 1.70-1.60 (m, 2H), 1.46 (s, 9H).

[000109] Synthesis of trans-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-2-oxo-[1,3'-bipiperidine]-4'-carboxamide. To the solution of trans-tert-butyl 4'-carbamoyl-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-2-oxo-[1,3'-bipiperidine]-1'-carboxylate 12 (60 mg, 0.11 mmol) in CH2Cl2 (1.0 mL) was added CF3CO2H (1.0 mL) at room temperature. The reaction mixture was stirred at room temperature for two hours, concentrated in vacuo to give the desired product 13 (43 mg, 90%) which was used directly in the next step without further purification. ESI-MS (M+H)+: 419.0.

[000110] Synthesis of trans-1'-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-2-oxo-[1,3 '-bipiperidine]-4'-carboxamide. To a solution of trans-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-2-oxo-[1,3'-bipiperidine]-4'-carboxamide 13 (42 mg, 0.10 mmol , 1.0 equiv) in 1-butanol (2 mL), 6-chloro-5-fluoropyrimidin-4-amine (18 mg, 0.12 mmol, 1.2 equiv) was added DIPEA (26 mg, 0.20 mmol, 2.0 equiv). The reaction solution was stirred at 120°C for 16 hours. The mixture was diluted with EtOAc (20 mL), washed with H2O (10 mL) and brine (10 mL), dried (Na2SO4), filtered and concentrated in vacuo. The crude product was purified by pre-HPLC (MeOH/H 2 O with 0.05% TFA as mobile phase) to give the compound (trans)-1'-(6-amino-5-fluoropyrimidin-4-yl)-3- ((3-chloro-5-(trifluoromethyl)phenyl)amino)-2-oxo-[1,3'-bipiperidine]-4'-carboxamide 14 (44 mg, yield: 83%) as a yellow solid. ESI-MS (M+H) +: 530.0. HPLC: (214 nm: 100%, 254 nm: 100%). 1H NMR (400 MHz, CD3OD) δ: 7.97 (s, 1H), 6.84 (s, 1H), 6.81 (s, 1H), 6.76 (s, 1H), 4.58- 4.52 (m, 2H), 4.09-4.03 (m, 1H), 3.52-3.35 (m, 3H), 3.29-3.27 (m, 4H), 3. 12-3.05 (m, 1H), 2.24-2.17 (m, 1H), 2.02-1.91 (m, 3H), 1.80-1.63 (m, 2H).

[000111] (3R,3'R,4'S)-1'-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-2- oxo-[1,3'-bipiperidine]-4'-carboxamide. The mixture of four diastereomers of compound 14 was separated into three peaks by SFC (IA (2 x 15 cm), 30% EtOH (0.1% DEA)/CO2, 10000 KPa (100 bar), 60 ml/min) and the title compound corresponded to peak 3. LCMS (Agilent460, 254 nm): ES (+) MS m/e = 530.1 (M+1) @ 1.20 min. 1H NMR (400 MHz, DMSO-d6) δ: 7.77 (d, J = 2.01 Hz, 1H), 7.38 (br.s., 1H), 6.94 (s, 2H), 6 .75 - 6.87 (m, 2H), 6.41 - 6.66 (m, 3H), 4.29 (br.s., 1H), 4.23 (d, J = 13.05 Hz, 1H), 3.96 - 4.18 (m, 2H), 3.44 (td, J = 6.15, 12.30 Hz, 1H), 3.24 - 3.33 (m, 1H), 3 .10 (br.s., 1H), 2.88 (br.s., 1H), 2.82 (t, J = 12.30 Hz, 1H), 2.13 (qd, J = 5.94 , 12.30 Hz, 1H), 1.74 - 1.93 (m, 3H), 1.58 - 1.74 (m, 1H), 1.41 - 1.58 (m, 1H).

[000112] (3S,3'R,4'S)-1'-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-2- oxo-[1,3'-bipiperidine]-4'-carboxamide. The mixture of four diastereomers of compound 14 was separated into three peaks by SFC (IA (2 x 15 cm), 30% EtOH (0.1% DEA)/CO2, 10000 KPa (100 bar), 60 ml/min) and the title compound corresponded to peak 2. LCMS (Agilent460, 254 nm): ES (+) MS m/e = 530.1 (M+1) @ 1.19 min. 1H NMR (400 MHz, DMSO-d6) δ: 7.77 (d, J = 1.76 Hz, 1H), 7.39 (br.s., 1H), 6.98 (s, 1H), 6 .96 (s, 1H), 6.72 - 6.88 (m, 2H), 6.57 (s, 2H), 6.54 (d, J = 7.78 Hz, 1H), 4.05 - 4.33 (m, 4H), 3.37 (t, J = 6.27 Hz, 2H), 3.11 (br.s., 1H), 2.94 (br.s., 1H), 2 .82 (t, J = 12.30 Hz, 1H), 2.02 - 2.16 (m, 1H), 1.75 - 1.92 (m, 3H), 1.57 - 1.74 (m , 1H), 1.36 - 1.54 (m, 1H).

[000113] (3S,3'S,4'R)-1'-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-2- oxo-[1,3'-bipiperidine]-4'-carboxamide. The mixture of four diastereomers of compound 14 was separated into three peaks by SFC (IA(2 x 15 cm), 30% EtOH (0.1% DEA)/CO 2 , 10000 KPa (100 bar), 60 ml/min). Peak 1 of 3 was further purified by SFC (AD-H (2 x 15cm), 30% iPrOH (0.1% DEA)/CO2, 10000 KPa (100 bar), 60 ml/min) to give the title compound . LCMS (Agilent 460, 254 nm): ES (+) MS m/e = 530.1 (M+1) @ 1.20 min. 1H NMR (400 MHz, DMSO-d6) δ: 7.77 (d, J = 1.76 Hz, 1H), 7.38 (br.s., 1H), 6.94 (s, 2H), 6 .83 (s, 1H), 6.80 (s, 1H), 6.42 - 6.66 (m, 3H), 4.18 - 4.47 (m, 2H), 3.95 - 4.18 (m, 2H), 3.39 - 3.52 (m, 1H), 3.24 - 3.31 (m, 1H), 3.10 (br.s., 1H), 2.88 (br. s., 1H), 2.82 (t, J = 12.30 Hz, 1H), 2.13 (qd, J = 5.91, 12.39 Hz, 1H), 1.73 - 1.92 ( m, 3H), 1.58 - 1.73 (m, 1H), 1.42 - 1.58 (m, 1H).

[000114] (3R,3'S,4'R)-1'-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-2- oxo-[1,3'-bipiperidine]-4'-carboxamide. The mixture of four diastereomers of compound 14 was separated into three peaks by SFC (IA(2 x 15 cm), 30% EtOH (0.1% DEA)/CO 2 , 10000 KPa (100 bar), 60 ml/min). Peak 1 of 3 was further purified by SFC (AD-H (2 x 15 cm), 30% iPrOH (0.1% DEA)/CO 2 , 10000 KPa (100 bar), 60 ml/min) to give the title compound. title. LCMS (Agilent 460, 254 nm): ES (+) MS m/e = 530.1 (M+1) @ 1.20 min. 1H NMR (400 MHz, DMSO-d6) δ: 7.77 (d, J = 1.76 Hz, 1H), 7.39 (br.s., 1H), 6.98 (s, 1H), 6 .96 (s, 1H), 6.73 - 6.88 (m, 2H), 6.57 (s, 2H), 6.54 (d, J = 7.78 Hz, 1H), 4.05 - 4.35 (m, 4H), 3.37 (t, J = 6.15 Hz, 2H), 3.12 (br.s., 1H), 2.94 (br.s., 1H), 2 .82 (t, J = 12.30 Hz, 1H), 2.09 (sxt, J = 5.80 Hz, 1H), 1.74 - 1.92 (m, 3H), 1.56 - 1, 73 (m, 1H), 1.36 - 1.52 (m, 1H). Example 3 Alternative synthesis of (3R,3'R,4'S)-1'-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)- 2-oxo-[1,3'-bipiperidine]-4'-carboxamide.

[000115] In addition to the methods described in Example 2, (3R,3'R,4'S)-1'-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl) )phenyl)amino)-2-oxo[1,3'-bipiperidine]-4'-carboxamide (compound I-1) was also synthesized according to Scheme 6. Scheme 6

[000116] 1-tert-Butyl 4-ethyl 3-Oxopiperidine-1,4-dicarboxylate 3-2. To a solution of 3-1 (5.0 kg, 19.1 mol, 1.0 equiv) in EtOH (50 L) under N2 was added (Boc)2O (4.2 kg, 19.1 mol, 1. 0 equiv), Et 3 N (1.9 kg, 19.1 mol, 1.0 equiv) and 10% Pd(OH) 2 /C (250 g, 10% w/w). After emptying and refilling with hydrogenation three times, the mixture was stirred under 1 atm of hydrogen at 50°C for 15 hours. LC-MS fully indicated consumption of 3-1. After the mixture had cooled to room temperature, the catalyst was filtered through a pad of celite and washed with EtOH (2.5 L). The filtrate was concentrated in vacuo to give a crude 3-2 (5.2 kg) as an oil, which was used in the next step without further purification.

[000117] (S)-1-tert-butyl 4-ethyl 3-((1-Phenylethyl)amino)-5,6-dihydropyridine-1,4(2H)-dicarboxylate (3-3). To a 100 L reactor equipped with a Dean-Stark apparatus was charged toluene (20 L), crude compound 3-2 (5.2 kg, 19.1 mol, 1.0 equiv) washed with toluene (30 L) , pTSA (329 g, 0.2 mol, 0.01 equiv), and S-(-)-α-methylbenzylamine .95 kg, 16.2 mol, 0.85 equiv). The mixture was heated to reflux with a blanket of nitrogen and the water removed through Dean-Stark. After 18 hours, LC-MS indicated complete consumption of 3-2. The mixture was then cooled to room temperature. The insolubles were removed by filtration, and the filtrate was concentrated in vacuo to dryness to give a crude 3-3 as a thick oil. This crude product was used in the next step without further purification. 3-10% amide by-product formed in this reaction and its structure was tentatively assigned based on LC-MS data.

[000118] (3R)-1-tert-butyl 4-ethyl 3-(((S)-1-phenylethyl)amino)piperidine-1,4-dicarboxylate (3-4). To a 100 L reactor loaded with NaBH4 (1.16 kg, 30.5 mol, 2.0 equiv) and anhydrous THF (60 L) under nitrogen was added TFA (10.5 kg, 92 mol, 6.0 equiv. ) slowly for 30 min while maintaining the temperature at 0~5°C. The mixture was then cooled to -45°C. In a separation reactor, crude product 3-3 was dissolved in anhydrous acetonitrile (30 L), which was slowly added to the above NaBH4/TFA solution while maintaining the internal temperature between -45~-30°C. The mixture was stirred at -45°C for one hour, after which HPLC indicated complete consumption of compound 3-3. The mixture was slowly diluted with ice water (50 kg) and the mixture was then heated to 10°C. The product was extracted with EtOAc (2 x 40 L) and the combined organic layers were washed with saturated NaHCO3 solution (20 L). The pH of the aqueous solution was ~8. The organic layers were dried (Na 2 SO 4 ) and concentrated in vacuo to dryness to give a residue which was later azeotroped with MeOH (10 L x 3) to remove excess EtOAc. In the end, a 10 L solution of crude 3-4 in MeOH was obtained, which was used directly in the subsequent step without further purification. ESI-MS (M+H-1) +: 377.2. 1H NMR (400 MHz, CD3OD) δ: 7.317.22 (m, 5H), 4.20 (q, 2H), 4.11-3.86 (m, 3H), 3.15 (s, 1H), 3.00-2.90 (m, 2H), 2.64 (d, 2H), 1.87-1.85 (m, 1H), 1.68 (s, 1H), 1.50-1, 25 (m, 15H).

[000119] (3R)-1-(tert-Butoxycarbonyl)-3-(((S)-1-phenylethyl)amino)piperidine-4-carboxylic acid (3-5). A 100 L reactor was charged with THF/MeOH (1:1, 80 L), a solution of LOH-H2O (2.5 kg, 60 mol, 4.0 equiv) in water (10 L) was added and a solution of crude 3-4 in MeOH (10 L) from above step. The resulting mixture was stirred at 22°C for 18 hours, at which time LC/MS indicated complete consumption of starting material 3-4. The solution was diluted with MTBE (40 L) and stirred for 20 minutes. The aqueous layer was separated, cooled to 0°C and neutralized with 3N HCl solution to a pH between 7-8, while keeping the internal temperature below 10°C. The solution was washed with DCM (5 x 30 L) or until LC/MS indicated no 3-5 product remaining in the aqueous layer. The combined organic layers were concentrated in vacuo to dryness, suspended in EtOAc and petroleum ether (2:1, 10 L) and stirred for two hours, the solids were filtered, washed with petroleum ether (5 L) and dried under vacuum. at 50°C for 18 hours to give the product (3.5 kg, 53% yield) as a 95% pure solid. Compound 3-5 is a mixture of ~30:70 trans/cis at C-4 and ~93:7 R:S at C-3. The average overall 3-1 yield is 43-55%. ESI-MS (M+H-1) +: 349.2. 1H NMR (400 MHz, CD3OD) δ: 8.22-8.06 (m, 5H) 4.11 (m, 1H), 3.86-3.82 (m, 1H), 3.59-3, 56 (m, 1H), 2.79-2.65 (m, 1H), 3.22-2.62 (m, 2H), 2.06-2.16 (m, 12H).

[000120] (3R)-1-(6-Amino-5-fluoropyrimidin-4-yl)-3-(((S)-1-phenylethyl)amino)piperidine-4-carboxylic acid (3-7). A 50 L reactor was charged with 10 L of 2N HCl and 3-5 (850 g, 2.44 mol, 1.0 equiv). The mixture was heated to 30°C and stirred for two hours, at which time HPLC indicated complete consumption of the initial 3-5. The solution was diluted with MTBE (4 L) and stirred for 20 min, the layers were separated and to the aqueous layer was added solid K2CO3 (660 g) over one hour to pH ~7. Additional K 2 CO 3 (660 g, 4.8 mol, 2.0 equiv) was added followed by 6-chloro-5-fluoropyrimidine-4-ylamine (360 g, 2.44 mol, 1.0 equiv) and 1,4- dioxane (5 L). The mixture was heated to gently reflux to 100°C and stirred at this temperature for 16 hours. HPLC indicated <2% of compound 3-6 remaining. The mixture was washed with DCM (2 x 5 L) and the organic wash solutions were discarded. The aqueous layer was treated with activated carbon (425 g) and the slurry was stirred for one hour at 30°C followed by filtration through diatomite. This activated carbon treatment was repeated. The resulting aqueous solution was neutralized to pH ~7 with concentrated HCl, and stirred at 22°C for 3 hours, the resulting slurry was filtered and the wet cake was washed with 1,4-dioxane/water (1:1, 1 .2 L), dried under vacuum at 50°C for 18 hours until KF ~0.5% of product 3-7 was obtained as a pale white solid (690 g, 81% yield) in 98.6% purity . The product contains a 1:9 mixture of cis/trans isomers at the C3 and C4 positions. ESI-MS (M+H-1) +: 460.2. 1H NMR (400 MHz, CD3OD) δ: 8.49 (d, J = 2.01 Hz, 1H), 8.218.14 (m, 5H) 4.94-4.90 (m, 1H), 4.63 (d, J = 11.55 Hz, 1H), 4.42 (m, 1H), 4.03 (m, 2H), 3.59 - 3.72 (m, 3H), 2.84 - 2, 93 (m, 1H), 2.20 - 2.31 (m, 1H), 2.15 (d, J = 6.78 Hz, 3H).

[000121] (3R)-3-Amino-1-(6-amino-5-fluoropyrimidin-4-yl)piperidine-4-carboxylic acid (3-8). A 10 L reaction was charged under N2, i-PrOH (3.5 L), H2O (3.5 L), 3-7 (1.0 equiv, 0.97 mol, 350 g), potassium fluoride mono -hydrate (290 g, 3.0 eq, 3.0 mol) and 35 g of 20% Pd(OH) 2 /C (10% v/w). After emptying/refilling with hydrogen three times, the mixture was heated to 40-50°C and vigorously stirred at that temperature under an atmosphere of hydrogen. After 18 hours, LC/MS indicated <1% starting material 3-7 remaining. The mixture was purged with N2 for 20 min, cooled to 22°C, and filtered. The wet cake and the filtrate contained the product and were processed separately.

[000122] The filtrate was concentrated in vacuo at 50°C to a volume of ~200 mL. After cooling to 20°C and stirring at this temperature for two hours, a slurry was obtained, and the solid was filtered, washed with water (400 mL) and dried under vacuum and at 50°C to give product 3-8 ( 65 g). The wet cake from the reaction filtration was stirred in 1N HCl (1 L) for 2 hours to dissolve the product and the remaining solid catalyst was then removed by filtration. The acidic filtrate was neutralized with solid LiOH to pH ~7 to precipitate product 3-8. The product was washed with water (200 mL), dried under vacuum and at 50°C to give 120 g of product. A total of 185 g of product was obtained in 98.7% purity in 75% yield based on H NMR. All source liquids were combined and concentrated to a volume of ~400 mL, resulting in a slurry, filtration, water washing, and drying yielded an additional 64 g solid of ~50% purity. 1H NMR (400 MHz, D2O): δ 7.77 (s, 1H) 4.12 (d, J = 14.05 Hz, 1H), 4.01 (d, J = 13.05 Hz, 1H), 3.26 (d, J = 13.80 Hz.1), 2.99 - 3.10 (m, 1H), 2.64 - 2.73 (m, 1H), 1.98 (dd, J = 3.39, 14.18 Hz, 1H), 1.74 - 1.87 (m, 1H).

[000123] ((3R,3'R)-1'-(tert-Butoxycarbonyl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-2-oxo-[1,3'- bipiperidine]-4'-carboxylic acid (3-10) To a solution of 3-8 (440 g， 1.9 mol, 1.0 equiv) in DMSO (10 L) was added 3-8A sequentially (640 g , 1.9 mol, 1.0 equiv) and sodium triacetoxyborohydride (STAB, 402.0 g, 3.8 mol, 2 equiv) and Et 3 N (190 g, 1.9 mol, 1.0 equiv). was heated to 50°C and stirred for 3 hours to show complete conversion by HPLC to intermediate 3-9.

[000124] The solution was diluted with MeOH (182 g, 5.7 mol, 3.0 eq.) to quench excess STAB, and the reaction was heated to 70~80°C. After 16 hours, HPLC indicated 22% product 3-10 formed and 61% intermediate 3-9 remaining and chiral HPLC indicated ~3% lactam epimer. The mixture was held at 70-80°C for an additional 24 hours to generate 50% 3-10, 35% 3-9, and 7% lactam epimer. After another 40 hours of stirring, 80% of 3-10 formed, 4% of 3-9 remained, and the lactam epimer increased to 14%. The mixture was cooled to 22°C, and quenched with a 2N NH4Cl solution (5 L) to generate a slurry mixture. After 30 minutes of stirring, the mixture was filtered and the wet cake was washed with water (3 L), dried under vacuum and at 55°C until KF<0.1. Crude 3-10 was obtained as a brown solid (850 g, 97.7%); chiral HPLC indicated 12.5% lactam epimer. This product was used directly without further purification.

[000125] (3R,3'R,4'S)-1'-(6-Amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-2 Acid -oxo-[1,3'-bipiperidine]-4'-carboxylic acid (3-10-trans). A 10 L reactor was charged under N2 with 3-10 (850 g, 1.9 mol, 1.0 equiv) in DMF (4.25 L, 5 v/p) to generate a clear solution was added 4-dimethylaminopyridine (DMAP 116g, 0.95 mol, 0.5 equiv) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI, 36.5 g, 0.19 mol, 0.1 equiv). After the mixture was stirred at 15 to 22°C for about one hour, additional EDCI (36.5 g, 0.19 mol, 0.1 equiv) was added and stirred for another one hour. HPLC indicated a 69:1 trans/cis mixture. The 3-10-trans product was not isolated and was converted to compound I-1 in a single step. 1H NMR (300 MHz, DMSO d6): δ 1.47-1.55 (m, 1H), 1.63-1.68 (m, 1H), 1.81-1.87 (m, 1H), 1.90-1.97 (m, 1H), 2.93-3.19 (m, 1H), 3.16-3.23 (m, 1H), 3.33-3.45 (m, 2H ) 4.07-4.33 (m, 3H), 6.80 (m, 1H), 6.94-6.98 (m, 1H), 7.107.16 (m, 2H), 7.91 (s , 1H).

[000126] (3R,3'R,4'S)-1'-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-2- oxo-[1,3'-bipiperidine]-4'-carboxamide (I-1). The above reaction mixture was charged at 22°C with O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU, 600 g, 1.9 mol, 1. 0 equiv), N,N-diisopropylethylamine (DIPEA, 1.0 kg, 9.5 mol, 5.0 equiv) and finally NH 4 Cl (260 g, 5.7 mol, 3.0 equiv). The resulting mixture was stirred at 15°C for one hour, HPLC indicated complete consumption of 3-10-trans, the mixture was poured into brine (25 L) and extracted with EtOAc (2 x 2L). The combined organic compounds were washed with brine (2 x 2L) and concentrated in vacuo below 45°C until dry to give crude I-1, which was purified by chromatography with EtOAc/petroleum ether/MeOH (1:1: 0 to 50:50:10) to generate three fractions, which contained 316 g, 98.8% chemical purity and 10.8% epimer, 160 g, 82.3% chemical purity and 17.5% epimer and 180 g, 61% purity and 11.3% epimer, respectively. The first two reactions above were combined and further purified by prep-HPLC to give 200 g of product with >99% purity and <1% epimer. 1H NMR (400 MHz, DMSO d6): δ 1.48-1.53 (m, 1H), 1.66-1.69 (m, 1H), 1.77-1.79 (m, 3H), 2.11-2.16 (m, 1H), 2.80-2.88 (m, 2H), 3.11 (s, 1H), 3.42-3.48 (m, 1H), 4. 0-4.25 (m, 4H), 6.58 (s, 3H), 6.80-6.85 (d, J = 10.2, 2H), 6.95 (s, 2H), 7, 40 (s, 1H), 7.77 (s, 1H). Example 4 Alternative synthesis of (3R,3'R,4'S)-1'-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)- 2-oxo-[1,3'-bipiperidine]-4'-carboxamide.

[000127] In addition to the methods described in Examples 2 and 3, (3R,3'R,4'S)-1'-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5- (trifluoromethyl)phenyl)amino)-2-oxo-[1,3'-bipiperidine]-4'-carboxamide (compound I-1) was also synthesized according to Scheme 6. Scheme 7

[000128] (3R)-3-Amino-1-(tert-butoxycarbonyl)piperidine-4-carboxylic acid (4-L-6). A 10 L reactor was charged under nitrogen with compound 4-5 (100 g, 0.287 mol), MeOH (6 L, 60 v/w) and 10 g of 20% Pd(OH) 2 /C. The reactor was evacuated/refilled with hydrogen three times and the mixture was heated to 40-50°C while stirring under 3 MPa of hydrogen for 40 hours. LC/MS indicated complete consumption of starting material 4-5. The mixture was cooled to 22°C and filtered, and the filtrate was concentrated in vacuo to dryness to give a solid product. This crude product was slurried in EtOH (500 mL) at 22°C for two hours, filtered and dried under vacuum at 50°C to give 85% yield of product 4-L-6 (60 g, 0.245 mol ) as a white solid.

[000129] (3R)-1-(tert-Butoxycarbonyl)-3-(((R)-4-((3-chloro-5-(trifluoromethyl)phenyl)amino)-5-ethoxy-5-oxopentyl) Acid amino)piperidine-4-carboxylic acid (4-L-8). To a solution of 4-L-6 (48.4 g, 0.197 mol) in DMSO (450 mL) was added Et3N (20.2 g, 0.199 mol, 1 equiv), 3-8A (67.4 g, 0.199 mol, 1 equiv) and sodium triacetoxyborohydride (STAB, 84.8 g, 0.40 mol, 2.0 equiv). The mixture was heated at 50°C for 30 min and stirred at that temperature for 3 hours. LC/MS indicated consumption of most of the 4-L-6 starting material and the formation of 4-L-8.

[000130] The reaction was stopped by the addition of EtOH (35 mL) and stirred at 50°C for 30 min. The mixture was heated at 75-85°C for 3 days. The mixture was cooled to 18°C and slowly transferred to water (6 L) while stirring vigorously to generate a slurry. After two hours, the solids were filtered and washed with water (3 x 3 L), dried under vacuum at 60-70°C for 24 hours to give 4-L-9 (114 g) as a brown solid. The solid was used directly in the subsequent step.

[000131] (3R,3'R,4'S)-1'-(tert-Butoxycarbonyl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-2-oxo-[1,3' Acid -bipiperidine]-4'-carboxylic acid (4-L-9-trans). To a solution of crude 4-L-9 (100 g) in DMF (500 mL) was added 4-dimethylaminopyridine (11 g, 0.09 mol, 0.5 equiv) and stirred at 20°C for 10 min. . 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (7.0 g, 0.036 mol, 0.2 equiv) was added and the reaction was stirred at 20°C for 3 hours. HPLC indicated a 57:43 cis/trans mixing ratio and additional EDAC (3.5 g, 0.018 mol, 0.1 equiv) was added. After 5 hours, HPLC indicated complete conversion to 4-L-9-trans. The mixture was transferred to water (2.25 L) slowly and the mixture was extracted with EtOAc (2 x 500 mL), and the organic layers were washed with brine (500 mL) and water (500 mL), concentrated in vacuo to dry to give crude 4-L-9-trans (100 g) as a brown solid. The crude product was dissolved in EtOAc (135 mL) at 60°C and then cooled to 20°C for one hour, followed by the addition of 50 mL of petroleum ether. The mixture was aged for two hours. The solids were filtered and washed with 3:1 EtOAc/petroleum ether (50 mL), dried under vacuum at 50°C for 16 hours to give 4-L-9-trans (23 g, 22% yield with 99 % purity). 1 H NMR (400 MHz, DMSO-d6) δ 6.94 (s, 2H), 6.81 (s, 1H), 6.54 - 6.61 (m, 1H), 3.99 - 4.08 (m, 1H), 3. 42 - 3.38 (m, 2H), 2.07 - 2.16 (m, 1H), 1.74 - 1.92 (m, 3H), 1 .39 (s, 9H).

[000132] (3R,3'R,4'S)-3-((3-Chloro-5-(trifluoromethyl)phenyl)amino)-2-oxo-[1,3'-bipiperidine]-4 Acid Hydrochloride '-carboxylic acid (4-L-10 trans). To a solution of 0.5N HCl in EtOAc (76 mL) was added 4-L-9 trans (20 g, 38 mmol) and heated at 20°C for 18 hours to generate a slurry. The solid was filtered, washed with EtOAc (5 mL) and dried under vacuum at 45°C for 18 hours to give 4-L-10 as the HCl salt (17 g, 97% yield).

[000133] (3R,3'R,4'S)-1'-(6-Amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-2 Acid -oxo-[1,3'-bipiperidine]-4'-carboxylic (3-10-trans). A solution of 4-L-10 (2.0 g, 4.38 mol), 6-chloro-5-fluoro-pyrimidin-4-ylamine (711 mg, 4.82 mmol, 1.1 equiv), DIPEA ( 1.52 mL, 8.77 mol, 2 eq.) in 40 mL of nBuOH was heated at 130-140°C for 72 hours. The mixture was cooled to 22°C and concentrated in vacuo to give a residue which was purified by column to give 3-10-trans (1.1 g, 47%). A relatively minor amount of (3S,3'R,4'S)-1'-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl) acid epimer amino)-2-oxo-[1,3'-bipiperidine]-4'-carboxylic acid was also observed. Intermediate 3-10-trans can be converted to compound I-1 by the procedure described above.

[000134] Compound I-1 was also synthesized according to Scheme 8.

[000135] The synthesis of (R)-tert-butyl 4-e 1-(3-chloro-5-(trifluoromethyl)phenyl)-5-oxopyrrolidine-2-carboxylate was synthesized using a similar procedure as in Phillips, D.P. ; Zhu, X.-F.; Lau, T.L.; Yang, K.; Liu, H. Tetrahedron Letters, 2009, 50, 7293 , whereby (S)-methyl 5-oxopyrrolidine-2-carboxylate and 1-chloro-4-iodobenzene were replaced by (S)-5-oxopyrrolidine-2-carboxylate ( R)-tert-butyl and 1-chloro-3-iodo-5-(trifluoromethyl)benzene.

[000136] (2R)-tert-Butyl 1-(3-chloro-5-(trifluoromethyl)phenyl)-5-hydroxypyrrolidine-2-carboxylate. An anhydrous solution of 4-e (11 g, 30 mmol) in Me-THF (100 mL) was cooled to -35°C under an atmosphere of nitrogen. A solution of DIABL-H (5.9 g, 42 mmol) in toluene (42 mL) was added dropwise while maintaining the temperature at -35°C. The reaction was monitored by HPLC and upon completion a solution of 1N Rochell's salt (100 mL) was added while keeping the reaction temperature below 0°C. The organic phase was separated, washed with 1N Rochell's salt (50 mL x 3) and separated, diluted with Et3N (4 mL), dried (Na2SO4) and concentrated in vacuo to give the 4-f (8.3 g) as an oil.

[000137] (3R)-1-(6-Amino-5-fluoropyrimidin-4-yl)-3-(((R)-5-(tert-butoxy)-4-((3-chloro-5- (trifluoromethyl)phenyl)amino)-5-oxopentyl)amino)piperidine-4-carboxylic acid. A solution of 4-f (37.4 g, 0.146 mmol) in DMF (700 mL) was treated with 3-8 (40.2 g, 0.11 mmol), Et3N (10.1 g, 0.1 mmol ) STAB (42.4 g, 0.2 mmol) and the mixture was heated at 55°C for 5 hours. The reaction was diluted with water (2.5 L), extracted with EtOAc (500 mL x 3), the organic phases were combined and washed with brine, separated, dried (Na2SO4) and concentrated in vacuo to give the 4-a ( 40.2 g) as a solid which was used without further purification.

[000138] (3R)-1-(6-Amino-5-fluoropyrimidin-4-yl)-3-(((R)-4-carboxy-4-((3-chloro-5-(trifluoromethyl)phenyl) acid )amino)butyl)amino)piperidine-4-carboxylic acid. To a 5N HCl solution (250 mL) was added 4-a t-butyl ester and the suspension was heated at 55°C for 5 hours while hydrolysis was monitored by HPLC. Upon completion of product formation, the water was removed in vacuo, resulting in a solid 4-b which was dried under vacuum and used without further purification.

[000139] (3R,3'R)-1'-(6-Amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-2-oxo acid -[1,3'-bipiperidine]-4'-carboxylic acid. To a solution of 4-b acid (55 g, 0.1 mol) in DMF (500 mL) was added a DIEA (64.5 g, 0.5 mol), CDI (32.5 g, 0.2 mol ) at 0°C. The solution was stirred for 1.5 h at 0°C, diluted with water (3 L), adjusted to pH 3 with HCl and extracted with EtOAc (2 L x 3). The organic phases were combined, dried (Na2SO4) and concentrated in vacuo to give the 4-c (48g).

[000140] The remaining steps for compound I-1 are completed using the procedures described above. Example 5 Synthesis of trans 3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-4'-(methylcarbamoyl)-2-oxo-[1,3'-bipiperidine]-1'-carboxylate -butyl.

[000141] Synthesis of trans-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-4'-(methylcarbamoyl)-2-oxo-[1,3'-bipiperidine]-1'-carboxylate tert-butyl. A similar procedure was used as described for the synthesis of (3'R,4'S)-1'-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl) phenyl)amino)-2-oxo-[1,3'-bipiperidine]-4'-carboxamide 14, to give the crude material which was purified by pre-HPLC (MeOH/H2O with 0.05% NH3.H2O as phase mobile) to give the title compound (360 mg, yield: 67%) as a yellow solid. ESI-MS (M+H) +: 544.18. HPLC: (214 nm: 100.0%, 254 nm: 100.0%). 1H NMR (400 MHz, CD3OD) (mixture of isomers) δ: 7.69-7.68 (m, 1H), 6.78 (s, 1H), 6.75 (s, 1H), 6.71 ( s, 1H), 4.39-4.36 (m, 2H), 4.09-4.03 (m, 1H), 3.53-3.31 (m, 3H), 3.20-3, 10 (m, 1H), 2.99-2.92 (m, 1H), 2.55 (s, 3H), 2.28-2.19 (m, 1H), 1.96-1.77 ( m, 5H), 1.68-1.58 (m, 1H).

[000142] (3R,3'R,4'S)-1'-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-N- methyl-2-oxo-[1,3'-bipiperidine]-4'-carboxamide. The mixture of four diastereomers was separated into two peaks by SFC (AD-H (2 x 15 cm), 50% 1:1 IPA:methanol (0.1% DEA)/CO2, 10000 KPa (100 bar), 60 ml /min). Peak 2 was further purified by SFC separation (AD-H (2 x 15 cm), 30% iPrOH(0.15% DEA)/CO2, 10000 KPa (100 bar), 60 ml/min) to give the title compound. title. LCMS (Agilent 460, 254 nm): ES (+) MS m/e = 544.1 (M+1) @ 1.24 min. 1H NMR (400 MHz, DMSO-d6) δ: 7.72 - 7.85 (m, 2H), 6.92 (s, 2H), 6.81 (s, 1H), 6.43 - 6.64 (m, 3H), 4.34 (br.s., 1H), 4.23 (d, J = 13.05 Hz, 1H), 3.93 - 4.19 (m, 2H), 3.37 - 3.49 (m, 1H), 3.22 - 3.30 (m, 1H), 3.13 (br.s., 1H), 2.84 (t, J = 12.05 Hz, 2H) , 2.57 (d, J = 4.52 Hz, 3H), 2.13 (qd, J = 6.05, 12.45 Hz, 1H), 1.60 - 1.89 (m, 4H), 1.40 - 1.58 (m, 1H).

[000143] (3S,3'R,4'S)-1'-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-N- methyl-2-oxo-[1,3'-bipiperidine]-4'-carboxamide. The mixture of four diastereomers was separated into two peaks by SFC (AD-H (2 x 15 cm), 50% 1:1 IPA:methanol (0.1% DEA)/CO2, 10000 KPa (100 bar), 60 ml /min). Peak 2 was further purified by SFC separation (AD-H (2 x 15 cm), 30% iPrOH(0.15% DEA)/CO2, 10000 KPa (100 bar), 60 ml/min) to give the title compound. title. LCMS (Agilent 460, 254 nm): ES (+) MS m/e = 544.1 (M+1) @ 1.24 min. 1H NMR (400 MHz, DMSO-d6) δ: 7.83 (q, J = 4.60 Hz, 1H), 7.77 (d, J = 1.76 Hz, 1H), 6.97 (d, J = 6.78 Hz, 2H), 6.81 (s, 1H), 6.58 (s, 2H), 6.53 (d, J = 7.78 Hz, 1H), 4.23 (d, J = 13.05 Hz, 2H), 3.90 - 4.19 (m, 2H), 3.14 (br.s., 1H), 2.92 (br.s., 1H), 2.74 - 2.90 (m, 1H), 2.55 (d, J = 4.52 Hz, 3H), 2.00 - 2.18 (m, 1H), 1.74 - 1.89 (m, 3H ), 1.56 - 1.74 (m, 1H), 1.34 - 1.50 (m, 1H).

[000144] (3S,3'S,4'R)-1'-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-N- methyl-2-oxo-[1,3'-bipiperidine]-4'-carboxamide. The mixture of four diastereomers was separated into two peaks by SFC (AD-H (2 x 15 cm), 50% 1:1 IPA:methanol (0.1% DEA)/CO2, 10000 KPa (100 bar), 60 ml /min). Peak 1 was further purified by SFC (AD-H (2 x 15 cm), 30% MeOH (0.15% DEA)/CO2, 10000 KPa (100 bar), 60 ml/min) to give the title compound . LCMS (Agilent 460, 254nm): ES (+) MS m/e = 544.1 (M+1) @ 1.23 min. 1H NMR (400 MHz, DMSO-d6) δ: 7.72 - 7.85 (m, 2H), 6.92 (s, 2H), 6.81 (s, 1H), 6.57 (s, 2H ), 6.54 (d, J = 7.53 Hz, 1H), 4.23 (d, J = 13.30 Hz, 1H), 4.15 (dd, J = 3.26, 12.30 Hz , 1H), 4.08 (td, J = 7.06, 10.48 Hz, 1H), 3.36 - 3.47 (m, 1H), 3.23 - 3.30 (m, 1H), 3.13 (br.s., 1H), 2.84 (t, J = 11.80 Hz, 2H), 2.57 (d, J = 4.52 Hz, 3H), 2.13 (qd, J = 6.17, 12.61 Hz, 1H), 1.62 - 1.91 (m, 4H), 1.42 - 1.57 (m, 1H).

[000145] trans-1'-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-N-methyl-2-oxo- [1,3'-bipiperidine]-4'-carboxamide. The mixture of four diastereomers was separated into two peaks by SFC (AD-H (2 x 15 cm), 50% 1:1 IPA:methanol (0.1% DEA)/CO2, 10000 KPa (100 bar), 60 ml /min). Peak 1 was further purified by SFC (AD-H (2 x 15 cm), 30% MeOH (0.15% DEA)/CO2, 10000 KPa (100 bar), 60 ml/min) to give the title compound . LCMS (Agilent 460, 254 nm): ES (+) MS m/e = 544.1 (M+1) @ 1.23 min. LCMS (Agilent 460, 254 nm): ES (+) MS m/e = 544.1 (M+1) @ 1.24 min. 1H NMR (400 MHz, DMSO-d6) δ: 7.80 - 7.89 (m, 1H), 7.72 - 7.80 (m, 1H), 6.97 (d, J = 6.53 Hz , 2H), 6.81 (s, 1H), 6.58 (s, 2H), 6.53 (d, J = 7.78 Hz, 1H), 4.23 (d, J = 13.05 Hz , 2H), 3.89 - 4.19 (m, 2H), 3.13 (br.s., 1H), 2.74 - 3.02 (m, J = 12.42, 12.42 Hz, 2H), 2.55 (d, J = 4.52 Hz, 3H), 2.08 (qd, J = 5.97, 12.20 Hz, 1H), 1.81 (td, J = 6.24 , 12.36 Hz, 3H), 1.56 - 1.74 (m, 1H), 1.33 - 1.51 (m, 1H). Example 6

[000146] Synthesis of trans-1'-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-N,N-dimethyl-2- oxo-[1,3'-bipiperidine]-4'-carboxamide. A similar procedure was used as described for the synthesis of (3'R,4'S)-1'-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl) phenyl)amino)-2-oxo-[1,3'-bipiperidine]-4'-carboxamide 14, to give the crude material which was purified by pre-HPLC (MeOH/H 2 O with 0.05% TFA as mobile phase) to give the title compound (45 mg, yield: 90%) as a yellow solid. ESI-MS (M+H) +: 558.0. HPLC: (214 nm: 98.2%, 254 nm: 100.0%). 1H NMR (400 MHz, CD3OD) δ: 7.77 (s, 1H), 6.92-6.89 (m, 1H), 6.83-6.81 (m, 1H), 6.79 (s , 1H), 4.38-4.38 (m, 2H), 4.05-4.00 (m, 2H), 3.55-3.53 (m, 1H), 3.45-3.40 (m, 2H), 3.15-2.89 (m, 7H), 2.21-2.16 (m, 1H), 1.90-1.86 (m, 3H), 1.66-1 .56 (m, 2H).

[000147] (3R,3'R,4'S)-1'-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-N, N-dimethyl-2-oxo-[1,3'-bipiperidine]-4'-carboxamide. The title compound was obtained from the chiral separation of trans-1'-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-N ,N-Dimethyl-2-oxo-[1,3'-bipiperidine]-4'-carboxamide using a two-step chiral SFC separation. First, the mixture was separated into two peaks containing a mixture of two diastereomers ((3'R,4'S)-1'-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5 -(trifluoromethyl)phenyl)amino)-N,N-dimethyl-2-oxo-[1,3'-bipiperidine]-4'-carboxamide and (3'S,4'R)-1'-(6-amino-5 -fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-N,N-dimethyl-2-oxo-[1,3'-bipiperidine]-4'-carboxamide) using a ChiralPak IC column (2 x 15 cm, 30% methanol w/0.1 DEA) and then the resulting mixture containing a pair of isomers was separated further into the single enantiomers using a ChiralPak IA column (2 x 15 cm , 30% methanol w/0.1% DEA 10000 KPa (100 bar)). ESI-MS (M+H) +: 558.0 1H NMR (400 MHz, CDCl 3 ) δ: 7.92 (d, J = 1.76 Hz, 1H), 6.94 (s, 1H), 6, 72 (d, J = 7.78 Hz, 2H), 5.21 (d, J = 3.51 Hz, 1H), 4.70 (s, 2H), 4.45 (dd, J = 2.76 , 12.80 Hz, 2H), 4.17 - 4.32 (m, 1H), 3.64 - 3.80 (m, 2H), 3.44 - 3.58 (s, 3H), 3. 09 (s, 3H), 3.00 - 3.09 (m, 1H), 2.95 (s, 3H), 2.40 (dd, J = 5.52, 13.30 Hz, 1H), 1 .63 - 1.99 (m, 3H), 1.26 - 1.43 (m, 1H).

[000148] (3S,3'R,4'S)-1'-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-N, N-dimethyl-2-oxo-[1,3'-bipiperidine]-4'-carboxamide. The title compound was obtained from the chiral separation of trans-1'-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino) -N,N-dimethyl-2-oxo-[1,3'-bipiperidine]-4'-carboxamide using a two-step chiral SFC separation. First, the mixture was separated into two peaks containing a mixture of two diastereomers ((3'R,4'S)-1'-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5 -(trifluoromethyl)phenyl)amino)-N,N-dimethyl-2-oxo-[1,3'-bipiperidine]-4'-carboxamide and (3'S,4'R)-1'-(6-amino-5 -fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-N,N-dimethyl-2-oxo-[1,3'-bipiperidine]-4'-carboxamide) using a ChiralPak IC column (2 x 15 cm, 30% methanol w/0.1 DEA) and then the resulting mixture containing a pair of isomers was separated further into the single enantiomers using a ChiralPak IA column (2 x 15 cm , 30% methanol w/0.1% DEA 10000 KPa (100 bar)). ESI-MS (M+H) +: 558.0 1H NMR (400 MHz, (400 MHz, CDCl 3 ) δ: 7.93 (d, J = 1.26 Hz, 1H), 6.93 (s, 1H ), 6.69 (br.s., 2H), 5.06 (d, J = 4.27 Hz, 1H), 4.71 (s, 1H), 4.45 (d, J = 12.55 Hz, 2H), 4.16 - 4.26 (m, 1H), 3.66 - 3.81 (m, 2H), 3.54 - 3.64 (m, 1H), 3.40 - 3, 54 (m, 2H), 3.01 - 3.10 (m, 4H), 2.95 (s, 3H), 2.37 (dd, J = 5.27, 13.05 Hz, 1H), 1 .91 - 2.02 (m, 2H), 1.48 - 1.75 (m, 2H).

[000149] (3S,3'S,4'R)-1'-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-N, N-dimethyl-2-oxo-[1,3'-bipiperidine]-4'-carboxamide. The title compound was obtained from the chiral separation of trans-1'-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-N ,N-Dimethyl-2-oxo-[1,3'-bipiperidine]-4'-carboxamide using a two-step chiral SFC separation. First, the mixture was separated into two peaks containing a mixture of two diastereomers ((3'R,4'S)-1'-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5 -(trifluoromethyl)phenyl)amino)-N,N-dimethyl-2-oxo-[1,3'-bipiperidine]-4'-carboxamide and (3'S,4'R)-1'-(6-amino-5 -fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-N,N-dimethyl-2-oxo-[1,3'-bipiperidine]-4'-carboxamide) using a ChiralPak IC column (2 x 15 cm, 30% methanol w/0.1 DEA) and then the resulting mixture containing a pair of isomers was separated further into the single enantiomers using a ChiralPak IA column (2 x 15 cm , 30% methanol w/0.1% DEA 10000 KPa (100 bar)). ESI-MS (M+H) +: 558.0 1H NMR (400 MHz, CDCl 3 ) δ: 7.92 (d, J = 1.76 Hz, 1H), 6.94 (s, 1H), 6, 72 (d, J = 7.78 Hz, 2H), 5.21 (d, J = 3.51 Hz, 1H), 4.70 (s, 2H), 4.45 (dd, J = 2.76 , 12.80 Hz, 2H), 4.19 - 4.30 (m, 1H), 3.66 - 3.79 (m, 2H), 3.47 - 3.56 (m, 3H), 3. 09 (s, 3H), 2.97 - 3.07 (m, 1H), 2.95 (s, 3H), 2.40 (dd, J = 5.52, 13.30 Hz, 1H), 1 .81 - 1.97 (m, 3H), 1.73 (dd, J = 3.76, 12.80 Hz, 1H).

[000150] (3R,3'S,4'R)-1'-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-N, N-dimethyl-2-oxo-[1,3'-bipiperidine]-4'-carboxamide. The title compound was obtained from the chiral separation of trans-1'-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino) -N,N-dimethyl-2-oxo-[1,3'-bipiperidine]-4'-carboxamide using a two-step chiral SFC separation. First, the mixture was separated into two peaks containing a mixture of two diastereomers ((3'R,4'S)-1'-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5 -(trifluoromethyl)phenyl)amino)-N,N-dimethyl-2-oxo-[1,3'-bipiperidine]-4'-carboxamide and (3'S,4'R)-1'-(6-amino-5 -fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-N,N-dimethyl-2-oxo-[1,3'-bipiperidine]-4'-carboxamide) using a ChiralPak IC column (2 x 15 cm, 30% methanol w/0.1 DEA) and then a ChiralPak IA column (2 x 15 cm, 30% methanol w/0.1% DEA 10000 KPa (100 Pub)). ESI-MS (M+H) +: 558.0 1H NMR (400 MHz, (400 MHz, CDCl 3 ) δ: 7.93 (d, J = 1.26 Hz, 1H), 6.93 (s, 1H ), 6.69 (br.s., 2H), 5.06 (d, J = 4.27 Hz, 1H), 4.71 (s, 1H), 4.45 (d, J = 12.55 Hz, 2H), 4.16 - 4.26 (m, 1H), 3.66 - 3.81 (m, 2H), 3.54 - 3.64 (m, 1H), 3.40 - 3, 54 (m, 2H), 3.01 - 3.10 (m, 4H), 2.95 (s, 3H), 2.37 (dd, J = 5.27, 13.05 Hz, 1H), 1 .91 - 2.02 (m, 2H), 1.48 - 1.75 (m, 2H) Example 7

[000151] Synthesis of trans-1'-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-4'-(4-methylpiperazine- 1-carbonyl)-[1,3'-bipiperidin]-2-one. A similar procedure was used as described for the synthesis of trans-1'-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-2- oxo-[1,3'-bipiperidine]-4'-carboxamide 14 to give 23 which was purified by reverse phase HPLC (MeOH/H2O with 0.05% NH3.H2O as mobile phase) to give the title compound (100 mg, yield: 70%) as a yellow solid. ESI-MS (M+H) +: 613.24. 1H NMR (400 MHz, CDCl3) δ: 7.94 (s, 1H), 6.94 (s, 1H), 6.73-6.67 (m, 2H), 5.25-5.03 (m , 1H), 4.71 (s, 2H), 4.49-4.40 (m, 2H), 4.35-4.16 (m, 1H), 3.823.64 (m, 3H), 3. 62-3.41 (m, 6H), 3.08-2.97 (m, 1H), 2.52-2.34 (m, 3H), 2.30-2.25 (m, 2H), 2.20 (s, 3H), 1.85-1.64 (m, 2H), 1.72-1.64 (m, 2H), 1.491.31 (m, 1H).

[000152] (3R,3'R,4'S)-1'-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-4'-(4-methylpiperazine-1-carbonyl)-[1,3'-bipiperidin]-2-one. The title compound was obtained from the chiral separation of trans-1'-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-N ,N-Dimethyl-2-oxo-[1,3'-bipiperidine]-4'-carboxamide 23 using a two-step chiral SFC separation. First, the mixture was separated into two peaks containing a mixture of two diastereomers ((3'R,4'S)-1'-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5 -(trifluoromethyl)phenyl)amino)-N,N-dimethyl-2-oxo-[1,3'-bipiperidine]-4'-carboxamide and (3'S,4'R)-1'-(6-amino-5 -fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-N,N-dimethyl-2-oxo-[1,3'-bipiperidine]-4'-carboxamide) using a ChiralPak IC column (2 x 15 cm, 30% methanol w/0.1 DEA) and then the resulting mixture containing a pair of isomers was separated into two single enantiomers using a ChiralPak IA column (2 x 15 cm , 30% methanol w/0.1% DEA 10000 KPa (100 bar)). ESI-MS (M+H) +: 613.2 1H NMR (400 MHz, CD3OD) δ: 7.95 (s, 1H), 6.83 - 6.97 (m, 3H), 4.50 - 4 .68 (m, 2H), 4.28 - 4.40 (m, 1H), 3.66 - 3.91 (m, 8H), 3.30 - 3.52 (m, 4H), 3.14 (t, J = 12.42 Hz, 2H), 2.73 (br.s., 2H), 2.27 (dd, J = 5.65, 12.93 Hz, 1H), 1.83 - 2 .05 (m, 2H), 1.54 - 1.79 (m, 2H).

[000153] (3S,3'R,4'S)-1'-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-4'-(4-methylpiperazine-1-carbonyl)-[1,3'-bipiperidin]-2-one. The title compound was obtained from the chiral separation of trans-1'-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-N ,N-Dimethyl-2-oxo-[1,3'-bipiperidine]-4'-carboxamide 23 using a two-step chiral SFC separation. First, the mixture was separated into two peaks containing a mixture of two diastereomers ((3'R,4'S)-1'-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5 -(trifluoromethyl)phenyl)amino)-N,N-dimethyl-2-oxo-[1,3'-bipiperidine]-4'-carboxamide and (3'S,4'R)-1'-(6-amino-5 -fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-N,N-dimethyl-2-oxo-[1,3'-bipiperidine]-4'-carboxamide) using a ChiralPak IC column (2 x 15 cm, 30% methanol w/0.1 DEA) and then the resulting mixture containing a pair of isomers was separated further into the single enantiomers using a ChiralPak IA column (2 x 15 cm , 30% methanol w/0.1% DEA 10000 KPa (100 bar)). ESI-MS (M+H)+: 613.2. 1H NMR (400 MHz, CD3OD) δ: 7.91 - 7.99 (m, 1H), 6.84 - 6.97 (m, 3H), 4.57 (dd, J = 14.18, 19, 70 Hz, 2H), 4.34 (br.s., 1H), 3.42 - 3.53 (m, 1H), 3.37 (d, J = 1.51 Hz, 3H), 3.05 - 3.19 (m, 1H), 2.86 (t, J = 7.53 Hz, 1H), 2.68 - 2.78 (m, 2H), 2.26 (dd, J = 5.65 , 12.93 Hz, 1H), 1.82 - 2.03 (m, 3H), 1.55 - 1.79 (m, 2H).

[000154] (3S,3'S,4'R)-1'-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-4'-(4-methylpiperazine-1-carbonyl)-[1,3'-bipiperidin]-2-one. The title compound was obtained from the chiral separation of trans-1'-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-N ,N-Dimethyl-2-oxo-[1,3'-bipiperidine]-4'-carboxamide 23 using a two-step chiral SFC separation. First, the mixture was separated into two peaks containing a mixture of two diastereomers ((3'R,4'S)-1'-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5 -(trifluoromethyl)phenyl)amino)-N,N-dimethyl-2-oxo-[1,3'-bipiperidine]-4'-carboxamide and (3'S,4'R)-1'-(6-amino-5 -fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-N,N-dimethyl-2-oxo-[1,3'-bipiperidine]-4'-carboxamide) using a ChiralPak IC column (2 x 15 cm, 30% methanol w/0.1 DEA) and then the resulting mixture containing a pair of isomers was separated further into the single enantiomers using a ChiralPak IA column (2 x 15 cm , 30% methanol w/0.1% DEA 10000 KPa (100 bar)). ESI-MS (M+H)+: 613.2. 1H NMR (400 MHz, CD3OD) δ: 7.94 (s, 1H), 6.87 - 6.93 (m, 3H), 4.57 (dd, J = 14.18, 19.70 Hz, 1H ), 4.34 (br.s., 1H), 3.43 - 3.51 (m, 1H), 3.36 - 3.38 (m, 2H), 3.13 (t, J = 12, 30 Hz, 1H), 2.86 (t, J = 7.53 Hz, 1H), 2.73 (br.s., 1H), 2.26 (dd, J = 5.65, 12.93 Hz , 1H), 1.96 - 2.03 (m, 1H), 1.83 - 1.94 (m, 1H), 1.51 - 1.75 (m, 1H).

[000155] ((3R,3'S,4'R)-1'-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-4 '-(4-Methylpiperazine-1-carbonyl)-[1,3'-bipiperidin]-2-one The title compound was obtained from the chiral separation of trans-1'-(6-amino-5-fluoropyrimidin -4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-N,N-dimethyl-2-oxo-[1,3'-bipiperidine]-4'-carboxamide 23 using a Two-step chiral SFC separation First, the mixture was separated into two peaks containing a mixture of two diastereomers ((3'R,4'S)-1'-(6-amino-5-fluoropyrimidin-4-yl)-3- ((3-chloro-5-(trifluoromethyl)phenyl)amino)-N,N-dimethyl-2-oxo-[1,3'-bipiperidine]-4'-carboxamide and (3'S,4'R)-1'-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-N,N-dimethyl-2-oxo-[1,3'-bipiperidine]-4'-carboxamide) using a ChiralPak IC column (2 x 15 cm, 30% methanol w/0.1 DEA) and then each mixture containing a pair of isomers was separated further into the two single enantiomers using a column chira 1Pak IA (2 x 15 cm, 30% methanol w/0.1% DEA 10000 KPa (100 bar)). 1H NMR (400 MHz, CD3OD) δ: 7.96 (br.s., 1H), 6.90 (br.s., 1H), 6.76 (d, J = 10.04 Hz, 2H), 4.60 (t, J = 14.06 Hz, 2H), 4.19 - 4.32 (m, 1H), 3.67 - 3.78 (m, 1H), 3.43 - 3.54 ( m, 3H), 3.35 - 3.38 (m, 3H), 3.16 (t, J = 12.42 Hz, 1H), 2.86 (t, J = 7.40 Hz, 2H), 2.79 (s, 3H), 2.32 (dd, J = 5.02, 12.80 Hz, 1H), 1.89 - 2.07 (m, 4H), 1.62 - 1.76 ( m, 5H). Example 8

[000156] Synthesis of 1'-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-fluoro-5-(trifluoromethyl)phenyl)amino)-2-oxo-[1,3'- bipiperidine]-4'-carboxamide. A similar procedure was used as described for the synthesis of (3'R,4'S)-1'-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl) )phenyl)amino)-2-oxo-[1,3'-bipiperidine]-4'-carboxamide 14 to give the crude material which was purified by pre-HPLC (MeOH/H2O with 0.05% NH3.H2O as phase mobile) to give the title compound (175 mg, yield: 69%) as a yellow solid. ESI-MS (M+H)+: 514.19. HPLC: (214 nm: 96.13%, 254 nm: 96.53%). 1H NMR (400 MHz, CD3OD) δ: 7.79-7.78 (m, 1H), 6.76 (s, 1H), 6.63-6.54 (m, 2H), 4.41-4 .36 (m, 2H), 4.084.06 (m, 1H), 3.55-3.41 (m, 3H), 3.28-3.25 (m, 1H), 2.99-2.93 (m, 1H), 2.28-2.21 (m, 1H), 1.98-1.78 (m, 6H).

[000157] (3R,3'R,4'S)-1'-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-fluoro-5-(trifluoromethyl)phenyl)amino)-2- oxo-[1,3'-bipiperidine]-4'-carboxamide. The mixture of the four diastereomers was separated into three peaks by SFC (IA (3 x 15 cm), 30% EtOH (0.1% DEA)/CO2, 10000 KPa (100 bar), 70 ml/min) and peak 3 corresponded to the title compound. LCMS (Agilent 460, 254 nm): ES (+) MS m/e = 514.0 (M+1) @ 1.09 min. 1H NMR (400 MHz, CDCl3) δ: 7.91 (br.s., 1H), 6.66 (d, J = 8.53 Hz, 1H), 6.63 (s, 1H), 6.46 (d, J = 10.79 Hz, 1H), 6.12 (br.s., 1H), 5.47 (br.s., 1H), 5.16 (d, J = 3.51 Hz, 1H), 4.91 (br.s., 2H), 4.35 - 4.54 (m, 2H), 3.82 (td, J = 5.11, 10.60 Hz, 2H), 3, 51 - 3.60 (m, 1H), 3.34 - 3.48 (m, 3H), 2.96 (t, J = 12.30 Hz, 1H), 2.35 - 2.47 (m, 1H), 1.91 - 2.06 (m, 3H), 1.84 (dq, J = 3.89, 12.76 Hz, 1H), 1.48 - 1.62 (m, 1H).

[000158] (3S,3'R,4'S)-1'-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-fluoro-5-(trifluoromethyl)phenyl)amino)-2- oxo-[1,3'-bipiperidine]-4'-carboxamide. The mixture of the four diastereomers was separated into three peaks by SFC (IA(3 x 15 cm), 30% EtOH (0.1% DEA)/CO2, 10000 KPa (100 bar), 70 ml/min). Peak 2 of 3 corresponded to the desired compound. LCMS (Agilent 460, 254 nm): ES (+) MS m/e = 514.0 (M+1) @ 1.10 min. 1H NMR (400 MHz, DMSO-d6) δ: 7.77 (d, J = 1.76 Hz, 1H), 7.39 (s, 1H), 6.85 (s, 1H), 6.81 ( s, 1H), 6.74 (d, J = 12.30 Hz, 1H), 6.47 - 6.66 (m, 4H), 4.23 (d, J = 12.80 Hz, 2H), 3.90 - 4.18 (m, 2H), 3.34 - 3.46 (m, 2H), 3.12 (br.s., 1H), 2.94 (br.s., 1H), 2.82 (t, J = 12.42 Hz, 1H), 2.10 (qd, J = 5.75, 12.11 Hz, 1H), 1.74 - 1.92 (m, 3H), 1 .56 - 1.72 (m, 1H), 1.37 - 1.52 (m, 1H).

[000159] (3R,3'S,4'R)-1'-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-fluoro-5-(trifluoromethyl)phenyl)amino)-2- oxo-[1,3'-bipiperidine]-4'-carboxamide. The mixture of the four diastereomers was separated into three peaks by SFC (IA (3 x 15 cm), 30% EtOH (0.1% DEA)/CO2, 10000 KPa (100 bar), 70 ml/min). Peak 1 of 3 was further purified by SFC (AI (3 x 15 cm), 30% iPrOH (0.1% DEA)/CO2, 10000 KPa (100 bar), 70 ml/min) to give the title compound . LCMS (Agilent 460, 254 nm): ES (+) MS m/e = 514.0 (M+1) @ 1.10 min. 1H NMR (400 MHz, DMSO-d6) δ: 7.77 (d, 1.5 Hz, 1H), 7.39 (s., 1H), 6.85 (s, 1H), 6.81 (s. ., 1H), 6.74 (d, J = 12.30 Hz, 1H), 6.47 - 6.66 (m, 4H), 4.16 - 4.46 (m, 2H), 3.95 - 4.16 (m, 2H), 3.34 - 3.48 (m, 2H), 3.12 (br.s., 1H), 2.87 - 3.01 (m, 2H), 2. 82 (t, J = 12.30 Hz, 1H), 2.10 (qd, J = 5.75, 12.11 Hz, 1H), 1.74 - 1.92 (m, 3H), 1.54 - 1.74 (m, 1H), 1.35 - 1.52 (m, 1H).

[000160] (3S,3'R,4'S)-1'-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-fluoro-5-(trifluoromethyl)phenyl)amino)-2- oxo-[1,3'-bipiperidine]-4'-carboxamide. The mixture of the four diastereomers was separated into three peaks by SFC (IA (3 x 15 cm), 30% EtOH (0.1% DEA)/CO2, 10000 KPa (100 bar), 70 ml/min). Peak 1 of 3 was further purified by SFC (AI (3 x 15 cm), 30% iPrOH (0.1% DEA)/CO2, 10000 KPa (100 bar), 70 ml/min) to give the title compound . LCMS (Agilent 460, 254 nm): ES (+) MS m/e = 514.0 (M+1) @ 1.10 min. 1H NMR (400 MHz, DMSO-d6) δ: 7.77 (d, J = 1.76 Hz, 1H), 7.38 (br.s., 1H), 6.84 (s, 2H), 6 .70 (d, J = 12.30 Hz, 1H), 6.47 - 6.65 (m, 4H), 4.18 - 4.48 (m, 2H), 3.92 - 4.18 (m , 2H), 3.38 - 3.49 (m, 1H), 3.20 - 3.30 (m, 1H), 3.11 (br.s., 1H), 2.88 - 2.99 ( m, 1H), 2.83 (t, J = 12.30 Hz, 1H), 2.14 (qd, J = 6.03, 12.52 Hz, 1H), 1.74 - 1.92 (m , 3H), 1.59 - 1.74 (m, 1H), 1.41 - 1.58 (m, 1H). Example 9

[000161] Synthesis of (3'R,4'S)-1'-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-fluorophenyl)amino)-2-oxo-[ 1,3'-bipiperidine]-4'-carboxamide. A similar procedure was used as described for the synthesis of (3'R,4'S)-1'-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl) )phenyl)amino)-2-oxo-[1,3'-bipiperidine]-4'-carboxamide 14 to give the crude material which was purified by pre-HPLC (MeOH/H2O with 0.05% NH3.H2O as phase mobile) to give the title compound (141 mg, Y: 30%) as a white solid. ESI-MS (M+H) +: 479.9. HPLC: (214 nm: 100%, 254 nm: 100%). 1H NMR (400 MHz, DMSO d6) δ: 7.78-7.77 (m, 1H), 7.40-7.38 (m, 1H), 6.86-6.82 (m, 1H), 6.62-6.55 (m, 3H), 6.45-6.37 (m, 3H), 4.26-3.94 (m, 4H), 3.47-3.39 (m, 1H ), 3.20-3.03 (m, 2H), 2.90-2.78 (m, 2H), 2.18-2.04 (m, 1H), 1.86-1.34 (m , 5H).

[000162] (3R,3'R,4'S)-1'-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-fluorophenyl)amino)-2-oxo-[ 1,3'-bipiperidine]-4'-carboxamide. The mixture of the four diastereomers was separated into three peaks by SFC (IC (2 x 15 cm), 25% MeOH (0.1% DEA)/CO2, 10000 KPa (100 bar), 60 ml/min) to generate the compound of the title as peak 3, respectively. LCMS (Agilent 460, 254nm): ES (+) MS m/e = 480.0 (M+1) @ 1.01 min. 1H NMR (400 MHz, CDCl3) δ: 7.90 (br.s., 1H), 6.44 (d, J = 8.53 Hz, 1H), 6.40 (s, 1H), 6.30 (br.s., 1H), 6.23 (d, J = 11.04 Hz, 1H), 5.63 (br.s., 1H), 5.09 (br.s., 1H), 4 .94 (br.s., 2H), 4.45 (d, J = 12.80 Hz, 2H), 3.68 - 3.95 (m, 2H), 3.49 - 3.56 (m, 2H), 3.35 - 3.46 (m, 2H), 2.89 - 2.99 (m, 1H), 2.31 - 2.44 (m, 1H), 1.90 - 2.04 ( m, 3H), 1.76 - 1.89 (m, 1H), 1.49 - 1.61 (m, 1H).

[000163] (3S,3'R,4'S)-1'-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-fluorophenyl)amino)-2-oxo-[ 1,3'-bipiperidine]-4'-carboxamide. The mixture of the four diastereomers was separated into three peaks by SFC (IC (2 x 15 cm), 25% MeOH (0.1% DEA)/CO2, 10000 KPa (100 bar), 60 ml/min) to generate the compound of the title as peak 1, respectively. LCMS (Agilent 460, 254nm): ES (+) MS m/e = 480.0 (M+1) @ 1.01 min. 1H NMR (400 MHz, DMSO-d6) δ: 7.77 (d, J = 1.76 Hz, 1H), 7.39 (s., 1H), 6.81 (s, 1H), 6.57 (s, 3H), 6.46 (d, J = 12.30 Hz, 1H), 6.39 (dd, J = 1.76, 8.78 Hz, 1H), 6.34 (d, J = 7.53 Hz, 1H), 4.28 (br.s., 1H), 4.23 (d, J = 13.05 Hz, 1H), 4.13 (dd, J = 2.76, 12, 30 Hz, 1H), 4.03 (td, J = 6.56, 10.98 Hz, 1H), 3.33 - 3.46 (m, 2H), 3.11 (br.s., 1H) , 2.94 (br.s., 1H), 2.82 (t, J = 12.30 Hz, 1H), 2.09 (qd, J = 5.75, 12.11 Hz, 1H), 1 .72 - 1.92 (m, 3H), 1.56 - 1.72 (m, 1H), 1.29 - 1.50 (m, 1H).

[000164] (3S,3'S,4'R)-1'-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-fluorophenyl)amino)-2-oxo-[ 1,3'-bipiperidine]-4'-carboxamide. The mixture of the four diastereomers was separated into three peaks by SFC (IC (2 x 15 cm), 25% MeOH(0.1% DEA)/CO2, 10000 KPa (100 bar), 60 ml/min). Peak 2 of 3 was further purified by SFC (AI (3 x 15 cm), 30% iPrOH (0.1% DEA)/CO2, 10000 KPa (100 bar), 60 ml/min) to give the title compound . LCMS (Agilent460, 254 nm): ES (+) MS m/e = 480.0 (M+1) @ 1.01 min. 1H NMR (400 MHz, DMSO-d6) δ: 7.77 (d, J = 2.01 Hz, 1H), 7.37 (br.s., 1H), 6.84 (s, 1H), 6 .57 (s, 2H), 6.55 (br.s., 1H), 6.30 - 6.48 (m, 3H), 4.28 (br.s., 1H), 4.23 (d , J = 13.05 Hz, 1H), 4.13 (dd, J = 3.39, 12.67 Hz, 1H), 3.97 (td, J = 6.84, 10.42 Hz, 1H) , 3.38 - 3.50 (m, 1H), 3.22 - 3.29 (m, 1H), 3.10 (br.s., 1H), 2.71-2.97 (m, 1H ), 2.83 (t, J = 12.30 Hz, 1H), 2.13 (qd, J = 6.13, 12.49 Hz, 1H), 1.73 - 1.91 (m, 3H) , 1.58 - 1.73 (m, 1H), 1.39 - 1.53 (m, 1H).

[000165] (3R,3'S,4'R)-1'-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-fluorophenyl)amino)-2-oxo-[ 1,3'-bipiperidine]-4'-carboxamide. The mixture of the four diastereomers was separated into three peaks by SFC (IC (2 x 15 cm), 25% MeOH(0.1% DEA)/CO2, 10000 KPa (100 bar), 60 ml/min). Peak 2 of 3 was further purified by SFC (AI (3 x 15 cm), 30% iPrOH (0.1% DEA)/CO2, 10000 KPa (100 bar), 60 ml/min) to give the title compound . LCMS (Agilent460, 254 nm): ES (+) MS m/e = 480.0 (M+1) @ 1.01 min. 1H NMR (400 MHz, DMSO-d6) δ 7.77 (d, J = 1.76 Hz, 1H), 7.39 (s, 1H), 6.81 (s, 1H), 6.57 (s , 3H), 6.46 (td, J = 2.01, 12.30 Hz, 1H), 6.39 (td, J = 1.95, 8.66 Hz, 1H), 6.34 (d, J = 7.53 Hz, 1H), 4.18 - 4.48 (m, 2H), 4.09 - 4.18 (m, 1H), 3.79 - 4.09 (m, 1H), 3 .33 - 3.45 (m, 2H), 3.11 (br.s., 1H), 2.92 (br.s., 1H), 2.82 (t, J = 12.42 Hz, 1H ), 2.09 (qd, J = 5.75, 12.11 Hz, 1H), 1.74 - 1.93 (m, 3H), 1.51 - 1.74 (m, 1H), 1. 32 - 1.51 (m, 1H). Example 10

[000166] Synthesis of (3'R,4'S)-1'-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethoxy)phenyl)amino)-2- oxo-[1,3'-bipiperidine]-4'-carboxamide. A similar procedure was used as described for the synthesis of (3'R,4'S)-1'-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl) )amino)-2-oxo-[1,3'-bipiperidine]-4'-carboxamide 14 to generate the crude material which was purified by pre-HPLC (MeOH/H2O with 0.05% NH3.H2O as mobile phase) to give the title compound (320 mg, yield: 44%) as a yellow solid. ESI-MS (M+H)+: 546.16. HPLC: (214 nm: 98.4%, 254 nm: 98.0%). 1H NMR (400 MHz, DMSO-d6) δ: 7.77 (s, 1H), 7.39 (s, 1H), 6.84-6.81 (m, 1H), 6.75-6.72 (m, 1H), 6.626.57 (m, 3H), 6.52-6.47 (m, 2H), 4.24-3.98 (m, 4H), 3.47-3.40 (m, 1H). , 1H), 3.17-2.99 (m, 2H), 2.86-2.79 (m, 2H), 2.15-1.99 (m, 1H), 1.86-1.60 (m, 4H), 1.48-1.39 (m, 1H).

[000167] (3R,3'S,4'R)-1'-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethoxy)phenyl)amino)-2- oxo-[1,3'-bipiperidine]-4'-carboxamide. The mixture of the four diastereomers was purified by SFC (AD-H(2 x 25 cm), 30% EtOH(0.1% DEA)/CO2, 10000 KPa (100 bar), 70 ml/min) to give the compound of title. LCMS (Agilent460, 254 nm): ES (+) MS m/e = 546.0 (M+1) @ 1.20 min. 1H NMR (400 MHz, DMSO-d6) δ: 7.77 (d, J = 1.76 Hz, 1H), 7.39 (br.s., 1H), 6.81 (s, 1H), 6 .75 (s, 1H), 6.62 (s, 1H), 6.57 (s, 2H), 6.38 - 6.52 (m, 2H), 4.23 (d, J = 13.05 Hz, 2H), 3.92 - 4.18 (m, 2H), 3.34 - 3.45 (m, 2H), 3.11 (br.s., 1H), 2.93 (br.s. ., 1H), 2.82 (t, J = 12.30 Hz, 1H), 2.08 (qd, J = 5.75, 12.11 Hz, 1H), 1.74 - 1.92 (m , 3H), 1.56 - 1.73 (m,

[000168] (3S,3'S,4'R)-1'-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethoxy)phenyl)amino)-2- oxo-[1,3'-bipiperidine]-4'-carboxamide. The mixture of the four diastereomers was purified by SFC (AD-H (2 x 25 cm), 30% EtOH (0.1% DEA)/CO2, 10000 KPa (100 bar), 70 ml/min) to give the compound of title. LCMS (Agilent 460, 254 nm): ES (+) MS m/e = 546.0 (M+1) @ 1.23 min. 1H NMR (400 MHz, DMSO-d6) δ: 7.77 (d, J = 2.01 Hz, 1H), 7.37 (br.s., 1H), 6.84 (s, 1H), 6 .72 (s, 1H), 6.60 (s, 1H), 6.57 (s, 2H), 6.43 - 6.54 (m, 2H), 4.23 (d, J = 13.05 Hz, 1H), 4.22 (br.s., 1H), 4.13 (dd, J = 3.26, 12.30 Hz, 1H), 4.01 (td, J = 6.81, 10 .23 Hz, 1H), 3.44 (td, J = 6.18, 12.49 Hz, 1H), 3.20 - 3.29 (m, 1H), 3.11 (br.s., 1H ), 2.88 (br.s., 1H), 2.83 (t, J = 12.30 Hz, 1H), 2.12 (qd, J = 6.05, 12.46 Hz, 1H), 1.73 - 1.91 (m, 3H), 1.59 - 1.73 (m, 1H), 1.48 (td, J = 9.41, 19.33 Hz, 1H).

[000169] (3S,3'R,4'S)-1'-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethoxy)phenyl)amino)-2- oxo-[1,3'-bipiperidine]-4'-carboxamide. The mixture of the four diastereomers was purified by SFC (AD-H (2 x 25 cm), 30% EtOH (0.1% DEA)/CO2, 10000 KPa (100 bar), 70 ml/min) to give the title compound. title. LCMS (Agilent 460, 254 nm): ES (+) MS m/e = 546.0 (M+1) @ 1.22 min. 1H NMR (400 MHz, DMSO-d6) δ: 7.77 (d, J = 2.01 Hz, 1H), 7.38 (br.s., 1H), 6.81 (s, 1H), 6 .75 (s, 1H), 6.62 (s, 1H), 6.57 (s, 2H), 6.42 - 6.51 (m, 2H), 4.23 (d, J = 12.80 Hz, 2H), 3.97 - 4.18 (m, 2H), 3.34 - 3.44 (m, 2H), 3.10 (br.s., 1H), 2.93 (br.s. ., 1H), 2.82 (t, J = 12.17 Hz, 1H), 2.03 - 2.15 (m, 1H), 1.77 - 1.90 (m, 3H), 1.57 - 1.73 (m, 1H), 1.37 - 1.48 (m, 1H).

[000170] (3R,3'R,4'S)-1'-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethoxy)phenyl)amino)-2- oxo-[1,3'-bipiperidine]-4'-carboxamide. The mixture of the four diastereomers was purified by SFC (AD-H (2 x 25 cm), 30% EtOH(0.1% DEA)/CO2, 100bar, 70 ml/min) to give the title compound. LCMS(Agilent 460, 254nm): ES (+) MS m/e = 546.0 (M+1) @ 1.23 min. 1H NMR (400 MHz, CDCl3) δ: 7.93 (s, 1H), 6.60 (s, 1H), 6.52 (s, 1H), 6.35 (s, 1H), 5.85 ( br.s., 1H), 5.32 (br.s., 1H), 4.97 - 5.15 (m, 1H), 4.78 (br.s., 2H), 4.46 (d , J = 13.05 Hz, 2H), 3.67 - 3.87 (m, 2H), 3.34 - 3.60 (m, 4H), 2.99 (t, J = 12.17 Hz, 1H), 2.34 - 2.49 (m, 1H), 1.91 - 2.08 (m, 3H), 1.83 (dq, J = 3.76, 12.72 Hz, 1H), 1 .47 - 1.74 (m, 1H). Example 11

[000171] In vitro BTK kinase assay: BTK-POLYGAT-LS ASSAY. The purpose of the in vitro BTK assay was to determine the potency of the compound against BTK by measuring the IC50. Compound inhibition was measured after monitoring the amount of phosphorylation of a fluorescein-labeled polyGAT peptide (Invitrogen PV3611) in the presence of active BTK enzyme (Upstate 14-552), ATP, and inhibitor. The BTK kinase reaction was performed in a black 96-well plate (costar 3694). For a typical assay, a 24 uL aliquot of an ATP/peptide master mix (final concentration; 10 uM ATP, 100 nM polyGAT) in kinase buffer (10 mM Tris-HCl pH 7.5, 10 mM MgCl2, 200 uM Na 3 PO 4 , 5 mM DTT, 0.01% Triton X-100, and 0.2 mg/ml casein) was added to each well. Next, 1 µL of a 40X titration of the compound 4 times in 100% DMSO solvent was added, followed by the addition of 15 µL of the BTK enzyme mix in 1X kinase buffer (with a final concentration of 0.25 nM) . The assay was incubated for 30 minutes before being stopped with 28 µL of a 50 mM EDTA solution. Aliquots (5 µl) of the kinase reaction were transferred to a low volume white 384-well plate (Corning 3674), and 5 µl of a 2X detection buffer (Invitrogen PV3574, with 4 nM Tb-PY20 antibody, Invitrogen PV3552) was added. The plate was covered and incubated for 45 minutes at room temperature. Time resolved fluorescence (TRF) was measured in M5 Molecular Devices (332 nm excitation; 488 nm emission; 518 nm fluorescein emission). IC50 values were calculated using a four-parameter fit with 100% enzyme activity determined from the DMSO control and 0% activity from the EDTA control.

[000172] Selected compounds of formula I were tested and found to be active in the polyGAT assay. Compounds I-1, I-2, I-3, I-4, I-5 and I-7 generated IC50 values of 0.73 nM, 0.68 nM, 2.07 nM, 0.63 nM, 1.6 nM, and 1.2 nM respectively. Compound I-6 has an IC50 value of less than 1 nM. The comparator compound IC, shown below, produced an IC50 value of 2.0 nM.

[000173] a) Protocol Summary: PXR has been shown to be a primary nuclear receptor that mediates drug-induced CYP3A4 expression (Bertilsson G, et al.; Proc Natl Acad Sci U S A. 1998, Oct 13;95 (21):12208-13). Based on this CYP3A4 induction pathway, the cell-based PXR reporter gene assay is commonly used to screen new molecular entities (NMEs) at an early drug discovery stage for their potential to induce CYP3A4 (Luo G, et al., Drug Metab Dispos. 2002 Jul;30(7):795-804.) Studies were designed to assess the effect of novel molecular entities (NMEs) on human PXR activation in DPX2 cells. Cell lines stably transfected with the PXR nuclear receptor and corresponding response elements were seeded in 96-well plates. Twenty-four hours after seeding, the cells were treated with 6 different concentrations of NMEs in triplicate wells (see below), and the cells were then returned to the incubator for a further 24 hours. At the end of this incubation period, the number of viable cells/well was determined using a Promega Cell Titer Fluor cytotoxicity assay. After this assay, Promega's ONE-Glo was added to the same wells and the activity of the reporter gene was evaluated.

[000174] b) Test System: The test system consisted of the stably transformed DPX2 tumor cell line placed in 96-well microtiter plates. An expression vector carrying the PXR nuclear receptor plus the appropriate enhancers and promoters linked to the luciferase reporter gene were stably integrated into these tumor cell lines. Receptor activation was assessed by monitoring the activity of the reporter gene and comparing the results with cells treated with the vehicle. Positive controls consist of cells treated with 6 different concentrations (0.1, 0.5, 1, 5, 10, and 20 μM) of rifampicin. In this way, compounds that activate PXR can be easily and quickly identified. Once stably integrated cell lines were used, it is possible to observe a 3- to 70-fold receptor activation.

[000175] c) Data Processing and Receptor Activation Kinetics: Data processed using MS-Excel were calculated as the mean (n = 3) and CV % of the amount of PXR activation relative to vehicle treated cells in each of 6 different doses. All activation data were normalized to the number of viable cells/well. Results were also expressed as a percentage of the response generated by the appropriate positive control at a dose of 10 μM. EC50 and Emax values were derived for test compounds that generate receptor activation using non-linear regression of typical log dose-response curves (Prism V5.0c, GraphPad Software, San Diego, CA). Agents that exhibited atypical dose-response curves were not analyzed in this way.

[000176] New Molecular Entities (NMEs): Test compounds were tested at 0.05, 0.1, 0.5, 1, 2.5, and 10 μM

[000177] Selected compounds of formula I were tested in the PXR assay. Compounds I-1, I-2, I-3, I-4, and I-5 generated a % PXR induction (relative to 10 µM rifampin) of 62%, 42%, 47%, 67%, and 90%, respectively. The comparator compound IC, shown above, produced a % PXR induction of 95%. Example 13 Protocol for the FastPatch hERG Inhibition Assay:

[000178] The cardiac potassium channel, hERG, is responsible for a fast delayed rectifier current (IKr) in the human ventricle and inhibition of IKr is the most common cause of cardiac action potential prolongation by non-cardiac drugs (see, for example, Weirich and Antoni, Basic Res. Cardiol., 93, Suppl. 1, 125-32, 1998; Yap and Camm, Clin. Exp. Allergy, 29, Suppl. 3, 174-81, 1999). Longer action potential duration has been cited as a causative factor in QT interval prolongation that has been associated with a dangerous ventricular arrhythmia, torsade de pointes (Brown and Rampe, Pharmaceutical News, 7, 15-20, 2000).

[000179] The in vitro effects of the provided compounds were investigated on the hERG (human ether-à-go-go related gene) potassium channel current (a surrogate for IKr, the rapidly activating delayed rectifier cardiac potassium current) expressed in human embryonic kidney (HEK293) cells stably transfected with hERG cDNA. Cells were placed in HEPES-buffered physiological saline in a coated glass 96-well plate and loaded with appropriate amounts of test and control solutions for a duration of a 3 minute exposure at each concentration. Test compound was diluted in 0.3% DMSO. An automated parallel patch clamp system, QPatch HT (Sophion Bioscience A/S, Denmark), was used to assess at various concentrations (eg, 10 μM). IC50 values were estimated based on hERG inhibition data. The study was conducted at ChanTest (14656 Neo Parkway, Cleveland, OH). QPatch screening is further described by Janzen and Bernasconi (eds.), High Throughput Screening, Methods and Protocols, Second Edition, vol. 565, chapter 10, pg. 209-223, 2009.

[000180] Selected compounds of formula I were tested in the hERG assay. Compounds I-1, I-2, I-3, and I-4 generated hERG IC50s of 15.6 uM, 30 uM, 14.6 uM, and 13.7 uM, respectively. Compound I-5 shows no observable activity in the 10 uM hERG assay (no IC50 available). Compound I-6 shows little observable hERG activity (< 20% inhibition) at 10uM (no IC50 available). Compound I-7 shows hERG activity (65% inhibition) at 10uM (no IC50 available). Comparator compound IC, shown above, produced an hERG IC50 of 1.18 uM or activity (87% inhibition) at 10 uM. Example 14 GSH Retention in Human Liver Microsome: Protocol

[000181] Test compound (final concentration 10 uM) is incubated with both human and rat liver microsomes (final concentration 1 mg/mL), together with activating cofactors NADPH (final concentration 1 mM), potassium phosphate ( final concentration 100 mM pH 7.4), magnesium chloride (final concentration 3.3 mM) and the GSH retention agent (final concentration 5 mM). The incubation mixture is incubated for 60 min at 37°C and finished with ice-cold acetonitrile (equal volume as the incubation mixture) and the supernatants isolated. Supernatants are injected directly for LC/MS/MS analysis or dried under N2 and reconstituted in a water:acetonitrile (80:20) mixture prior to LC/MS/MS analysis. The corresponding GSH conjugate is evaluated by LC/MS/MS using a Qtof MSe TOF5600/Xevo Triple. Example 15 Rat Collagen-Induced Arthritis Model

[000182] Collagen-induced arthritis (CIA) model in female Lewis rats requires primary B and T cell immune responses for immunization of collagen type II (CII) for the development of a severe inflammatory disease (see Goldschmidt TJ, Holmdahl R. Cell Immunol. 154(1):240-8, 1994; Helfgott, S. M., et al.; Clin. Immunol. Immunopathol. 31:403, 1984; Holmdahl R. et al., J Autoimmun.7(6) ):739-52, 1994; and Stuart, J.M., et al., J. Exp. Med. 155:1, 1982). Clinical illness begins after a secondary IIC challenge and the disease progresses over the next eight days.

[000183] Generally, female Lewis rats are immunized with bovine type II collagen in incomplete Freund's adjuvant. Rats (N=10/group) receive daily oral administration of test compound or BID vehicle by oral gavage starting on day 1 (therapeutic). Clinical severity of arthritis is assessed by ankle caliper measurements taken every day beginning on Day 0.

[000184] Detailed protocol: Female Lewis rats are immunized subcutaneously with bovine type II collagen (1:1 emulsion of 2 mg/ml bovine CII in 0.01 N acetic acid: Incomplete Freund's Adjuvant) at three dorsal skin sites . Six days after immunization, mice received a second subcutaneous injection of bovine CII. A suspension compound or vehicle of formula I (0.5% CMC, 0.1% Tween 80) is administered by oral BID gavage starting on day 0 (prophylactic) (n=10 animals/group). Clinical severity of ASD is assessed by ankle caliper measurements taken every day beginning on Day 9. Baseline ankle caliper measurements are taken and confirmed as clinically normal (0.260-0.264 inch) for prophylactic treatment . Baseline ankle caliper measurements for animals with stabilized disease are evaluated on day 1 of therapeutic dosing and animals are randomly assigned to treatment groups after confirmation of clinical disease onset (0.2751-0.2755 inch ). Data are analyzed for all groups using a one-way analysis of variance (one-way ANOVA) together with an appropriate multiple comparison post-test. Significance for all tests is set at p < 0.05. Example 16 Analysis of BCR pathway activation through inhibition of PLCY2 phosphorylation

[000185] Protocol: One day before treatment, Ramos cells were plated at a density of 3x10 5 cells per well in 200 µL of complete medium in 96-well tissue culture filter plates (Millipore, Billerica, MA). On the day of treatment, the used medium was filtered off and the cells resuspended in 200 μL of serum-free medium containing serial dilutions of compound and 0.1% DMSO, then incubated for two hours at 37°C. Cells were stimulated for 5 minutes with 10 μg/mL of goat anti-human IgM at 37°C. All medium was filtered off and cells were washed with ice-cold PBS, then lysed on ice for 1 hour with lysis buffer containing: 20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 2 mM Na3VO4, 1% Triton X-100, 0.1% SDS, Protease Inhibitor Cocktail, 1 mM Phenylmethylsulfonyl Fluoride, (PMSF), Phosphatase 2 Inhibitor Mix (Sigma cat # P5726 from Sigma, St. Louis, Mo.), and phosphatase inhibitor 3 mix (Sigma cat # P0044 from Sigma, St. Louis, Mo.). Lysates were subsequently transferred to standard MSD plates (Meso Scale Discovery, (MSD), (Gaithersburg, Maryland)), pretreated with capture antibody (B10 anti-total PLCy2 antibody, (SantaCruz Biotechnologies (Santa Cruz, CA)) and blocked with BSA according to the manufacturer's instructions. Lysates were incubated on the prepared MSD plates overnight at 4°C with gentle shaking. The wells were washed three times with TBST and treated with anti pPLCy2 (SantaCruz) in BSA 1 % in PBS for one hour at room temperature. The wells were again washed three times with TBST and treated with sulfo-labeled anti-rabbit antibody (MSD) for one hour at room temperature. After washing with TBST, MSD Ready Buffer was added and luminescence is measured on a SECTOR Imager 6000 MSD. Maximal response is determined as mean luminescence in wells containing stimulated cells treated with anti-IgM and DMSO. Minimal response is determined as mean luminescence in wells s containing unstimulated cells treated with DMSO alone. The maximum and minimum values are used to normalize the luminescence in the compost treatment wells. Normalized values are plotted against compound concentration on a log scale, and then analyzed using Prizm software (GraphPad Software, Inc.). A sigmoid dose-response equation with variable slope is used to fit the data and generate a concentration of 50% inhibition (IC50).

[000186] Ramos cells were incubated in 96-well plates with a variety of concentrations of a compound of formula I for two hours, stimulated with 10 μg/mL of anti-IgM for 5 minutes, and PLCY2 phosphorylation was measured using an electrochemical luminescent immunoassay. EC50 is calculated using GraphPad Prism software. Example 17 Human B cell proliferation induced by BCR inhibition

[000187] Human CD19+ B cells are stimulated with an anti-IgM antibody and the activity of a compound of formula I is evaluated in terms of change in cellular metabolism after 72 hours. In this context, cellular metabolism directly correlates with cell activation and proliferation, and may also reflect cell survival during proliferation. Anti-IgM antibody is evaluated for effect on B cell proliferation and determined to exhibit a half maximum concentration for activation of 10 μg/ml. Using these activation conditions, varying concentrations of test compound, in triplicate in 0.1% DMSO, were evaluated for the impact on cellular metabolism of CD19+ B cells isolated from different donors.

[000188] Protocol: Human B cells were isolated from peripheral blood mononuclear cells or buffy coats using Ficoll-Hypaque gradients (Amersham) and negatively selected by magnetic cell sorting (Human B Cell Isolation Kit II, Miltenyi Biotec). Purity of target cells is determined by flow cytometry, staining for B cell, T cell and monocyte markers (CD19, CD3, CD14, respectively; BD Biosciences). Data were collected on a FACsCaliber flow cytometer and analyzed using FloJo software (BD Biosciences). The purity of human B cell preparations is routinely greater than 95%. Negatively selected human B cells were stimulated with 10 µg/ml anti-IgM F(ab')2 (Jackson ImmunoResearch) in 96-well plates. 100,000 B cells in 0.2 mL RPMI + 10% FBS were treated with varying concentrations (titrated from 5000 nM to 0 nM in 0.5% DMSO) of a compound of formula I in triplicate wells or vehicle control at final concentration of 0.5% DMSO for 30 minutes at 37°C, 5% CO2, then cells were stimulated with 10 μg/mL anti-IgM F(ab')2. B cells were stimulated for 72 hours at 37°C, 5% CO 2 . Proliferation was measured using the CellTiter-Glo reagent (Promega) as measured in a luminometer. Mean values were plotted against maximum proliferation and IC50 values were determined using GraphPad Prism v5 software. Example 18 Evaluation of the effect of compounds on activation of myeloid cells in vitro

[000189] FCYR activation of primary human macrophages. Autoantibody and immune complex-mediated activation through FcyR can be modeled by macrophage activation with immobilized IgG. Primary human macrophages derived from GM-CSF-treated monocytes upregulate the activation of markers such as CD80, CD86, MHC and FCYRIII receptor antigens. Macrophages derived from human monocytes can be activated by plaque-bound purified human IgG. This stimulus cross-links the FCYRIII receptor and induces the secretion of pro-inflammatory cytokines, such as TNFα, IL-6, IL1β and MCP-1. The compound of formula I was evaluated for inhibition of cytokine expression following FcR activation of human macrophages.

[000190] In general, macrophages were cultured on plates previously incubated with purified IgG and then washed. Test compound titers (10,000 nM to 0 nM) are added to these cultures. Cell culture supernatants were analyzed by ELISA for TNFα and IL-6 expression.

[000191] Protocol: Human monocytes were isolated from buffy coats of healthy donors and negatively selected by magnetic cell separation (Monocyte Isolation Kit II, Miltenyi Biotec). Purified monocytes were cultured in standard media supplemented with low IgG FBS and 100 ng/mL GM-CSF for 5-7 days to induce macrophage differentiation. Cultured macrophages were stimulated with 100 µg/ml of purified IgG bound to the plate ± one titer of test compound (10 µM to 0 nM). Supernatants were collected after 4 hours and 18 hours and analyzed for TNFα and IL-6, respectively. Example 19 Efficacy in Mouse Collagen Antibody-Induced Arthritis

[000192] This Example relates not only to arthritis, but also assesses the activity of autoantibodies and immune complexes in vivo and is therefore relevant to other inflammatory diseases such as SLE. In this experiment, the activity of autoantibodies and immune complexes produce a pathological parameter that is dependent on FcR signaling, and the Fc portion of these antibodies is inhibited by the administration of a compound of formula I.

[000193] The collagen antibody-induced arthritis (CAIA) model in female DBA/1 mice does not require cognate T and B cell responses for the induction of inflammation, but rather depends on immune effector mechanisms for the development of clinical disease. A cocktail of four anti-collagen II specific monoclonal antibodies (CII) and immune stimulatory lipopolysaccharide (LPS) administered 3 days after CII-specific antibody transfer promotes Fc receptor-antibody binding (Kagari T. et al.; J Immunol.170:4318-24 (2003)), immune complex formation, complement activation (NK Band, et al.; Clin Exp Immunol. 159:100-8 (2010)) and pro-inflammatory cytokine production to induce a severe inflammatory disease over a period of 10 days.

[000194] In general, arthritis is induced by injecting a cocktail of monoclonal anti-collagen antibodies into DBA/1 mice on day 0. Mice (N=10/group) receive daily oral administration of QD or BID test compound , as indicated, starting on day 0. Paw inflammation was assessed daily.

[000195] Protocol: 6-8 week old DBA/1 mice received 2 mg of a monoclonal antibody cocktail of four arthritogenic clones (Chondrex#10100) i.v. on day 0 followed by a 50 µg dose of LPS on the next 3 days. The test compound or vehicle suspension (0.5% CMC, 0.1% Tween 80) is administered BID by oral gavage, starting on day 0 (10 animals/group) immediately prior to i.v. of the antibody cocktail. Clinical severity of ASD was assessed by monitoring inflammation in all four paws, applying a scale ranging from 0 to 4. Each paw is rated as follows: 0, normal; 1, mild but definite redness and swelling of the ankle or wrist, or redness and swelling of any severity for 1 or 2 fingers; 2, moderate to severe redness and swelling of the ankle or wrist, or more than two fingers; 3, redness and swelling (pronounced edema) of the entire paw; and 4, maximally inflamed limb with involvement of several joints. The sum of the four individual scores is the arthritis index, with a maximum possible score of 16 for each animal.

Claims (2)

1. Use of a compound having formula I:

or a pharmaceutically acceptable salt thereof, wherein: each R1 is independently hydrogen, an optionally substituted C1-6 aliphatic group, an optionally substituted 3-7 membered monocyclic heterocyclic group, or a heterocyclylalkyl group having 3-7 optionally carbon atoms substituted and 1-3 heteroatoms independently selected from nitrogen, oxygen and sulfur; or two R1 groups are joined with their intermediate atoms forming an optionally substituted saturated or partially unsaturated 3-7 membered monocyclic heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen and sulfur; wherein optionally substituted groups may be substituted with halogen, -NO2, -CN, -OR, -SR, -N(R)2, -C(O)R, -CO2R, -N(R)C(O)OR , -C(O)N(R)2, -OC(O)R, -N(R)C(O)R, -S(O)R, -S(O)2R, or -S(O) 2N(R)2; each R is independently hydrogen or C1-6 aliphatic; or two R groups attached on the same nitrogen are joined with their intermediate atoms to form an optionally substituted, saturated or partially unsaturated 3-7 membered monocyclic heterocyclic ring having 1-2 heteroatoms, wherein any second heteroatom is independently selected from nitrogen, oxygen and sulfur; R2 Ring A is ;

R2 is -Cl or -F; and R3 is -CF3, -OCF3, or -F or a pharmaceutical composition thereof, characterized in that it is in the preparation of a medicament for treating a disorder, wherein the disorder is rheumatoid arthritis, lupus erythematosus, atopic dermatitis or cancer . 2. Use according to claim 1, characterized in that the cancer is leukemia or lymphoma.